CN106102725A - The cyclodextrin composite of the selective ATP inhibitor of encapsulating and application thereof - Google Patents

The cyclodextrin composite of the selective ATP inhibitor of encapsulating and application thereof Download PDF

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CN106102725A
CN106102725A CN201580013842.3A CN201580013842A CN106102725A CN 106102725 A CN106102725 A CN 106102725A CN 201580013842 A CN201580013842 A CN 201580013842A CN 106102725 A CN106102725 A CN 106102725A
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cyclodextrin
composition
cancer
brpa
cell
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J-F.格施温德
S.加纳帕蒂-坎尼亚庞
S.苏
B.沃格斯泰恩
K.W.金兹勒
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Johns Hopkins University
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Abstract

The present invention provides the composition comprising to encapsulate the cyclodextrin of selective ATP inhibitor, and its application.

Description

The cyclodextrin composite of the selective ATP inhibitor of encapsulating and application thereof
Related application
This application claims the U.S. Provisional Application No. 61/927,259 submitted on January 14th, 2014, and on May 13rd, 2014 submits to The senior interest of U.S. Provisional Application No. 61/992,572, it is incorporated herein by reference.
Rights statement
The present invention is under governmental support, at Grant No. R01 CA160771, P30 of being authorized by NIH Carry out under CA006973, and NCRR UL1 RR 025005.U.S. government has certain rights in the invention.This statement includes Observe 37 C.F.R. § 401.14 (a) (f) (4) interior being only and be not construed as disclosure and/or require only one The opinion of invention or accreditation.
Background of invention
The knowledge of glycolysis rather than oxidative phosphorylation that cancer cell depends on increase to survive is known as " Wo Baige hypothesis (Warburg hypothesis)” (Warburg (1956) Science123:309-314).This concept constitutes use sugar Basis (the Shaw (2006) of unique target that glycolysis and related enzyme thereof are developed as new anticancer therapeutic agentCurr. Opin. Cell Biol.18:598-608；Gatenby and Gillies (2007)Biochem. Cell Biol. 39:1358- 1366).One such medicine is 3-bromo acetone acid (3-BrPA), a kind of synthesis be used as irreversible glycolytic inhibitor The brominated derivative (Ko etc. (2001) of pyruvic acidCancer Lett.173:83-91；Geschwind etc. (2002)Cancer Res. 62:3909-3913).It is by targeting glycolytic ferment, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) affects energy Amount metabolism (Ganapathy-Kanniappan etc. (2009)AntiCancer Res.29:4909-4918).Additionally, 3-BrPA Antitumaous effect be all consistent for multiple tumor models in vitro and in vivo and repeatably.Various tumours by Prove that to 3-BrPA treatment be sensitive, including, for example, liver cancer (Geschwind etc. (2002)Cancer Res. 62: 3909-30913；Vali etc. (2007)J. Vasc. Interv. Radiol. 18:95-101；And Ganapathy- Kanniappan etc. (2012)Radiology262:834-845), cancer of pancreas (Cao etc. (2008)Clin. Cancer Res.14:1831-1839；Bhardwaj etc. (2010)AntiCancer Res.30:743-749；With (2013) such as OtaTarget Oncol.8:145-151), brain tumor (El Sayed etc. (2012)J. Bioenerg. Biomembr. 44:61- 79；Davidescu etc. (2012)J. Bioenerg. Biomembr.44:51-60) with breast cancer (Buijs etc. (2013)J. Vasc. Interv. Radiol. 24:737-743).In a word, GAPDH suppression and 3-BrPA molecular specificity Determine, be probably treatment cancer, particularly a feasible plan of solid malignant via 3-BrPA target tumor glycolysis Slightly (Ganapathy-Kanniappan etc. (2012)Oncotarget 3:940-95；Ganapathy-Kanniappan etc. (2013) AntiCancer Res. 33:13-20)。
Regardless of selective ATP inhibitor, the acid of such as 3-halogen acetone is as 3-BrPA is for the potentiality for the treatment of use, so And, there is several factors that the system of hampering gives the exploitation of preparation.For example, the alkylation of the acid of 3-halogen acetone and related compound (chemical) characteristic gives their extremely strong reactivity with electrophilic molecule, and this usually needs to increase dosage, and it has increase toxicity, Particularly increase the alkylating negative effect near injection site.Particularly, water or be typically found in protein any The existence of nucleophilic group, such as amino or sulfydryl, inactivates this compound in chemistry.Equally, such compound internal surely Qualitative included that the glutathione in blood and the circulatory system, NADH and other redox molecules are affected by multiple factors.Therefore, important , keep these compounds not affected by such factor, at least up to cycling through first.
While it is recognized that protective seletion ATP inhibitor, the acid of such as 3-halogen acetone is as 3-BrPA, until they are passed To organ or tissue for they system transmission under antitumous effects it is critical that, it is achieved the many of such protection Method, for example, encapsulate them, not yet successfully in liposome, microballoon, nanosphere, nano particle, vacuole etc..For example, as it is known that point Son such as 3-BrPA undesirably rapid ooze out from Pegylation (PEGylated) liposome or with protein such as albumin- Albumin reaction in base nano particle.Although fragmentary report has recorded intraperitoneal in preclinical models for the 3-BrPA and has passed Passing, curative effect and dosage are very limited amount of.Due to these failures, 3-BrPA therapy is withdrawn at present in regional area transmission (for example, percutaneous ablative, intra-arterial transmission, and intra-tumoral injection) rather than system transmission (Kunjithapatham etc. (2013)BMC Res. Notes 6: 277)。
Therefore, this area is suitable for selective ATP inhibitor such as 3-halogen acetone acid that system gives as 3-to identifying The composition of BrPA has very big demand.
Summary of the invention
The present invention is based in part on such discovery, will ATP produce selective depressant, such as 3-halogen acetone acid (example Such as 3-BrPA), it is encapsulated in cyclodextrin: a) by protection halogen moiety away from the aqueous and close of described compound ineffective will be made Nuclear environment, stablizes alkylating agent in vivo, and b) provides and maintain the steady of compound necessary to the reasonable half life of internal compound Fixed release.
On the one hand, provide and comprise cyclodextrin and the composition of medicine that below general formula represents:, Wherein, occur independently of one another: X represents halide, sulphonic acid ester, carboxylate, alkoxide or amine oxide；R1Represent OR, H, N (R”)2, C1-C6 alkyl, C6-C12 aryl, the miscellaneous alkyl of C1-C6, or C6-C12 heteroaryl；R " represents H, C1-C6 alkyl, or C6- C12 aryl；R represents H, alkali metal, C1-C6 alkyl, C6-C12 aryl or C (O) R '；With R ' represents H, C1-C20 alkyl or C6- C12 aryl, its cyclodextrin entrapped drug.In one embodiment, at least one α-D-glucopyranoside list of cyclodextrin At least one hydroxy chemical group of unit is substituted by ionogenic chemical group.In another embodiment, at least one α- At least one hydroxy chemical group of D-glycopyranoside units is selected from C2, C3 and C6 hydroxy chemical group.Implement at another In scheme, C2, C3 and C6 hydroxy chemical group ionogenicization of at least one α-D-glycopyranoside units of cyclodextrin Learn group to substitute.In a still further embodiment, at least one α-D-glycopyranoside units of cyclodextrin is selected from cyclodextrin 2nd, the 3rd, the 4th, the 5th, the 6th, the 7th, 8, and all of α-D-glycopyranoside units.In another embodiment, ionogenic chemistry base Group is identical in the position of all replacements.In still another embodiment, ionogenic chemical group is alkalescent functional group Or weakly acidic functional group.For example, alkalescent functional group (X) can have according to CH3-X-The pK between 6.5 and 8.5aOr weak acid Property functional group (Y) can have according to CH3The pK between 4.0 and 6.5 of-Ya.In a still further embodiment, alkalescent or weak Acidic functionality is selected from amino, second diaminourea, dimethyl second diaminourea, dimethyl benzene amino, dimethylnaphthalene amino, succinyl Base, carboxyl, sulfonyl, and Sulfuric acid functional groups.In another embodiment, cyclodextrin has the pK between 4.0 and 8.5a1。 In still another embodiment, composition is liquid or solid pharmaceutical preparation.In a still further embodiment, medicine is not charged Lotus or hydrophobic.In another embodiment, cyclodextrin is selected from beta-schardinger dextrin, alpha-cyclodextrin, and gamma-cyclodextrin.Again In one embodiment, cyclodextrin is beta-schardinger dextrin.In a still further embodiment, medicine is the acid of 3-halogen acetone.At another In individual embodiment, medicine is 3-bromo acetone acid.In still another embodiment, composition is formulated for system and gives. In a still further embodiment, composition also comprises anti-cancer therapeutic agent.
On the other hand, one is provided to comprise compositions described herein, and the kit of operation instructions.
It yet still another aspect, provide the method for the experimenter that a kind for the treatment of suffers from cancer, it includes that giving subject has The compositions described herein of effect amount.In one embodiment, composition gives through system.In another embodiment, System gives selected from oral, intravenous, intraperitoneal, subcutaneous, and intramuscular gives.In still another embodiment, experimenter is with extremely Few a kind of other anti-cancer therapy for treating.In a still further embodiment, at least one other anti-cancer therapy is that radiation is treated Method.In another embodiment, cancer is solid tumor.In still another embodiment, cancer is selected from liver cancer, cancer of pancreas, lung Cancer and breast cancer.In a still further embodiment, cancer is liver cancer.In another embodiment, experimenter is mammal. In still another embodiment, mammal is people.
Brief description
Fig. 1 shows the chemical constitution of beta-schardinger dextrin molecule and ring topologies (see such as Rasheed etc. (2008)Sci. Pharm. 76: 567-598)。
Fig. 2 A-2B display 3-BrPA or β-CD-3-BrPA after treatment 24 hours to MiaPaCa2 cell (Fig. 2 A) and The impact of Suit-2 cell (Fig. 2 B).
Fig. 3 shows the impact on MiaPaCa2 cell after treating 72 hours for 3-BrPA or the β-CD-3-BrPA.
Fig. 4 shows the impact on tumor growth in vivo for the β-CD-3-BrPA.
Fig. 5 is shown in the complete tumor response in 3 mouse of β-CD-3-BrPA treatment.
Fig. 6 shows such as the histopathological analysis result of the original position MiaPaCa-2 tumour treated in figures 4 and 5.
Fig. 7 shows the result of NMR spectroscopy.The illustration amplifying shows the high field displacement (0.1 ppm) of methene proton, this It is to observe after the complexing of β-CD-3-BrPA.
Fig. 8 is shown in MiaPaCa-2 (upper row) and Suit-2 (middle row) cell, according to based on luminous cellular activities What 2D and the 3D organotypic cell of power was cultivated kills curve.Cell is made to cultivate 72 hours under normal oxygen and anoxia condition, then cruelly Be exposed to 3-bromo acetone acid (3-BrPA), 1:1-beta-schardinger dextrin (CD)-3-BrPA, or the β-CD being only used as comparison to continue 24 little When.Make cell cultivate 24 hours, then process 72 hours with gemcitabine.(lower row) is cultivated for 3D organotypic cell,lucMiaPaCa-2 cell cultivates 6 days altogether under normal oxygen or anoxia condition.Exist by 3-BrPA or β-CD-3-BrPA single treatment Within 5th day, carry out, continue 24 hours.Become exposed to gemcitabine at the 3rd day 72 hours.Biodiversity resources was carried out at the 6th day To evaluate Medicated Permeation and the impact on Cells viability.Lower right box contains the immunity-trace to HIF-1 α to confirm that anoxic is deposited ?.
Fig. 9 shows effect in the cultivation of 3D organotypic cell for the 3-bromo acetone acid.Uniform embedded collagen I-matrixlucMiaPaCa-2 cell confirms (the 1st day) by confocal optics microscope.3D organotypic cell cultures is in normal oxygen condition Lower cultivate and every other day process with 3-BrPA, accumulative three times.(the latter is only used for for phase contrast microscopy and biodiversity resources MiaPaCa-2 cell) carried out at the 6th day, to evaluate the impact on cytomorphology and viablity.F-actin and cracking The immunofluorescence dyeing of Caspase-3 is carried out in the freezing microtome section of 3D organotypic cell cultures.It is multiple that DAPI is used as nucleic acid Stain.
Figure 10 shows further effect in the cultivation of 3D organotypic cell for the 3-bromo acetone acid.Be uniformly embedded into collagen I- The Suit-2 cell of matrix confirms (the 1st day) by confocal optics microscope.3D organotypic cell cultures is in normal oxygen condition Lower cultivate and every other day process with 3-BrPA, accumulative three times.(the latter is only used for for phase contrast microscopy and biodiversity resources MiaPaCa-2 cell) carried out at the 6th day, to evaluate the effect to cytomorphology and viablity.F-actin and cracking The immunofluorescence dyeing of Caspase-3 is carried out in the freezing microtome section that 3D organotypic cell is cultivated.It is multiple that DAPI is used as nucleic acid Stain.
Figure 11 A-11D shows the impact on cellular invasiveness for the 3-bromo acetone acid.MiaPaCa-2 (Figure 11 A) and Suit-2 (Figure 11 B) cell is inoculated in Boyden invasion and attack cell.Overnight incubation is followed by processed 48 hours by 3-bromo acetone acid (MiaPaCa-2) or 72 hours (Suit-2).The intrusion cell of the film bottom side of invasion and attack insert uses the dyeing of Ji's nurse Sa sample (Giemsa-like staining) dyes.Image shows by 4x, 10x, and the cell of the invasion and attack of 20x enlargement ratio.Invade By measurement, the area of staining cell in the full visual field of 10x calculates the Relative quantification attacked.MMP-9 activity and secretion exist In the concentrated supernatant of MiaPaCa-2 and Suit-2 cell, surveyed by zymography (Figure 11 C) and immunoblotting (Figure 11 D) Fixed.(*) conspicuousness statistically (p-value < 0.05) is represented.
The internal effect of Figure 12 A-12D display beta-schardinger dextrin-3-bromo acetone acid.With 1.5 x 10 altogether6IndividuallucMiaPaCa-2 cell in-situ implants 42 male nude mouses altogether.After xenograft growth 1 week, use biodiversity resources (BLI) tumour is confirmed.The animal of representative number is shown in Figure 12 A.Animal accept at random β-CD-3-BrPA (N=21), Free 3-BrPA (N=7), gemcitabine (N=7), and β-CD (N=7).Animal imaging weekly once, through the mistake of 28 days Journey.Signal macro-progress is shown in Figure 12 B.Analyze according to Kaplan-Meier, show with the animal of free 3-BrPA treatment The toxicity of excessive treatment-related, causes the loss of the animal of at the end of experiment 5/7, so that the related number of statistics is not Can survive and include in final graphical analysis (Figure 12 C).Medium-comparison (β-CD), when intraperitoneal gives, does not shows Any treatment-related toxicity and be inert (Figure 12 C).When completing experiment, put to death all of animal and explore Property autopsy, to extract organ and to evaluate potential infringement.For β-CD-3-BrPA, when comparing with inert agents (Figure 12 D), Do not observe organ toxicity's (function of organization).
Figure 13 shows in vitro pathology and immunohistochemistry tumor analysis.With β-CD, gemcitabine, or β-CD-3-BrPA The H&E dyeing of the tumour that (3 kinds of representational tumours are illustrated) is processed shows the therapeutic action of β-CD-3-BrPA.In H&E-dye The region that square in the complete-tumour general survey of look is designated as analyzing the Anti-tumor effect of medicine further and amplifies, it is by counterincision The Caspase-3 solving and the dyeing of Ki67 are confirmed.In addition, determine the GAPDH of the major target as 3-BrPA, with And the substantially reducing of MCT-1 as specific transport protein.
Detailed Description Of The Invention
Have determined that cyclodextrin can encapsulate selective depressant such as 3-halogen acetone acid (for example, 3-BrPA) that ATP produces herein, with Stablize alkylated compound in aqueous environments, and reduce nucleophilic entity in protein close to its ability, thus reduce Its system toxicity simultaneously maintains its alkylation ability.Herein in multiple body outer cell lines, use the cyclodextrin (example of multi-form Such as β and α) and activating agent packing relative to the different ratio of cyclodextrin, and in vivo animal tumor model confirms so Composition.For example, such composition has shown that at this selective depressant maintaining ATP to produce kills in vitro and in vivo The functional character of cancer cell, so that in order to system gives, their activity can be saved and protect until it arrives at target tissue, device Official, and/or tumour, reduce toxicity simultaneously to greatest extent.This mensuration is unexpected, because it is known that cyclodextrin is by directly Catalytic action, particularly increases pH, has the effect (Rasheed etc. (2008) stable to the destruction of chemical compound lotSci. Pharm.76: 567-598).Although having expected that this catalytic action of cyclodextrin is sour (because they are for 3-halogen acetone The halide derivative of pyruvic acid) it is big, determine that cyclodextrin is actually protected and stablizes 3-BrPA surprisingly.Also make us frightened Determine with being surprised, substitute on its α-D-glycopyranoside units one or more by the ionogen producing negative electrical charge (anion) One or more hydroxyl and the cyclodextrin modified, than those, there is the ring of the ionogenic group of generation positive charge (cation) Dextrin or the cyclodextrin of unmodified, the α-of such as unmodified or beta-schardinger dextrin preferably stablize the acid of 3-halogen acetone.It is not bound by opinion Constraint, it is believed that the anionicsite on cyclodextrin force halogen acetone acid (for example, 3-BrPA) halogen atom (for example, bromine) sit Fall in the cavities.Also determine surprisingly beta-schardinger dextrin encapsulating in protection with the 3-BrPA of form stablizing 3-BrPA, especially It is curative effect and also curative effect aspect is significantly better than alpha-cyclodextrin in vitro in vivo.
Therefore, the present invention provide comprise such 3-halogen acetone acid compound being encapsulated in cyclodextrin composition and Kit, and preparation and the method using such composition and kit.
A. define
In order to be easier to understand the present invention, some term and phrase below with entire disclosure defined in.
Article " one " and " one " are used to refer to one of object or more than (that is, an at least one) grammer pair herein As.For example, " key element " means a key element or more than a key element.
Term " 3-bromo acetone acid " or " 3-BrPA " refer to 3-bromo acetone acid, the analog of 3-bromo acetone acid and derivative Thing, the prodrug of 3-bromo acetone acid, the metabolite of 3-bromo acetone acid and salt thereof.
Term " giving " means to provide medicine or composition to experimenter, and includes, but not limited to by medical speciality people Member give and oneself-give.
Term " cancer " includes, but not limited to solid tumor and blood-born tumor.Term cancer includes skin, tissue, device The disease of official, bone, cartilage, blood and blood vessel.Term " cancer " also includes primary and metastatic cancer.
Term " suppression (inhibit) " or " suppression (inhibits) " mean to compare experimenter, cell, life with untreated Thing approach, or BA compares or and target, such as solid malignant in the experimenter before treatment experimenter Growth fraction relatively, reduce, suppress, weaken, weaken, stop, or the development of stable disease, illness or the patient's condition or progress, biology The growth of the activity of approach, or BA, such as solid malignant, for example, reaches at least 10%, the 20%th, the 30%th, the 40%th, 50%th, the 60%th, the 70%th, the 80%th, the 90%th, the 95%th, the 98%th, 99%, or even 100%.So-called term " reduction " means to suppress, suppresses, weakens, Weaken, stop, or stablize the symptom of Cancerous disease, illness or the patient's condition.It it is to be appreciated that, although be not excluded, but treatment disease, Illness or the patient's condition do not need described disease, illness, the patient's condition or associated symptom and fully eliminate.
Term " regulation " refers to the rise (that is, activate or stimulate) of reaction, lowers (that is, suppression or stop), or both group Close or separate.
Term " pharmaceutically acceptable " is used to refer to herein, in the range of rational medical judgment, be suitable for and people With the contact tissue of animal, and without excessive toxicity, excitant, allergic reaction, or other problem or complication, with rational profit Those compounds that benefit/Hazard ratio matches, material, composition, and/or formulation.
Term " pharmaceutically-acceptable carrier " is used to refer to pharmaceutically-acceptable material, composition or matchmaker herein It is situated between, such as liquid or solid filler, diluent, excipient, or solvent coating material, relate to carrying or transport motif compound From health organ, or part is to another organ of health, or part.Every kind of carrier is fitted at other compositions with preparation Join and to patient harmless in the sense that must be " acceptable ".Some can be used as the material of pharmaceutically-acceptable carrier Example includes: (1) sugar, such as lactose, dextrose plus saccharose；(2) starch, such as cornstarch and farina；(3) Cellulose, and its derivative, such as sodium carboxymethylcellulose, ethyl cellulose and cellulose acetate；(4) powdered tragacanth； (5) Fructus Hordei Germinatus；(6) gelatin；(7) talcum powder；(8) excipient, such as cocoa butter and suppository wax；(9) oil, such as peanut oil, Cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil and soybean oil；(10) glycol, such as propane diols；(11) polyalcohol, Such as glycerine, sorbierite, mannitol and polyethylene glycol；(12) ester, such as ethyl oleate and ethyl laurate；(13) agar； (14) buffer, such as magnesium hydroxide and aluminium hydroxide；(15) alginic acid；(16) apirogen water；(17) isotonic saline solution；(18) Ringer's mixture；(19) ethanol；(20) pH buffer solution；(21) polyester, Merlon and/or polyanhydride；(22) other are used Non-toxicity compatible substances in pharmaceutical preparation.
Term " pharmaceutically-acceptable salt " refers to the nothing-poison, inorganic and organic salt relatively of compound.
" experimenter " can include for goals of medicine, such as in order to treat existing disease, illness, the patient's condition or in order to prevent disease The preventative-therapeutic human experimenter of the generation of sick, illness or the patient's condition or for medical science, animal doctor's purpose, or development purpose is dynamic Thing experimenter.Suitable animal subjects includes that mammal includes, but not limited to primate, for example, people, monkey, ape, Gibbon, chimpanzee, orangutan, macaque etc.；Ox, for example, ox, bull etc.；Sheep, for example, sheep etc.；He-goat, for example, goat Deng；Pig, for example, piggy, boar etc.；Horse, for example, horse, donkey, zebra etc.；Cats, including wildcat and domestic cat；Canid, Including dog；Lagomorph, including rabbit, hare etc.；And rodent, including mouse, rat, cavy etc..Animal can be to turn base Because of animal.In some embodiments, experimenter is people, includes, but not limited to fetus, neonate, baby, teenager, and grows up Experimenter.Additionally, " experimenter " can include suffering from or suspect the patient suffering from disease, illness or the patient's condition.Therefore, term is " tested Person " and " patient " can exchange use at this.Experimenter also includes animal disease model (for example, rat or the mouse for experiment Deng).
Term " prevents ", " preventing ", " prevention ", " prophylactic treatment " etc. refer to that reducing experimenter occurs disease, illness or disease The possibility of condition, described experimenter does not suffer from, but is in the risk that disease, illness or the patient's condition occur or is susceptible to suffer from these diseases Disease, illness or the patient's condition.
Term " suspecting ill experimenter " means that experimenter shows one or more clinical indication of disease or illness. In certain embodiments, disease or illness are cancers.In certain embodiments, cancer is leukaemia or lymthoma.
Term " experimenter in need " means the identified experimenter needing and curing or treat.
Term " system gives ", " Formulations for systemic administration ", " periphery gives " and " peripherally administered " mean to give compound, medicine or Other materials (are not directly entered central nervous system), so that it enters the system of patient, thus stand metabolism and other classes Like process, for example, subcutaneous administration.
Term " therapeutic agent " or " medicine " refer to produce host the medicament of required biological action.Chemotherapeutics and gene Toxic agents is the example of therapeutic agent, and they are the materials of commonly known chemical, rather than biological agents, or lead to respectively Cross the specific mechanism of action and produce therapeutic action.The example of the therapeutic agent of biological origin includes growth factor, hormone, and cell The factor.Various therapeutic agents are known in the art and can be identified by their effect.Some therapeutic agent can regulate red blood cell Propagation and differentiation.Example includes chemotherapy nucleotides, medicine, hormone, non-specific (e.g., non-antibody) protein, oligonucleotides (for example, being incorporated into the ASON of target nucleic acid sequence (for example, mRNA sequence)), peptide, and peptidomimetic.
Term " therapeutic action " refers to animal, particularly mammal, and more particularly in people, is drawn by pharmacological active substance The local risen or systemic effect.Therefore this term refers to be intended in animal or people diagnosis, cures, alleviates, treats or prevent Disease or any material of the health needed for for improving or Mental development and state.Phrase " therapeutically effective amount " means to be suitable for In the rational interests/Hazard ratio of any treatment, produce the amount of this type of material of some required local or systemic effect.At certain In a little embodiments, the therapeutically effective amount of compound will depend upon which its therapeutic index, dissolubility etc..For example, by the side of the present invention The q.s that some compound of method discovery can produce the rational interests/Hazard ratio being applicable to so treat gives.
" therapeutically effective amount " and " effective dose " means compound, the material comprising the compounds of this invention as the term is employed herein Material or the amount of composition, it is efficiently used at least cell subsets of animal, to be applicable to the reasonable of any therapeutic treatment Interests/Hazard ratio produces some required therapeutic actions.
The disease of experimenter " treated " in term or " treatment " suffers from the experimenter of disease and instigate experimenter to stand drug therapy, For example, medicine is given, so that at least one symptom of described disease mitigates or prevents from deteriorating.
The exception of term " tumour ", " solid malignant " or " neoplasm " phalangeal cell or uncontrolled growth are formed Pathology.Preferably, tumour is malignant tumour, such as by the malignant tumour of formation of cancer.
B. cyclodextrin
Term " cyclodextrin " refers to the oligosacharides cyclic family being made up of 5 or more α-D-glycopyranoside units, described Glycosidic units is bonded together by the C1-C4 with ring topologies, wherein relatively big the and smaller opening of circulus Some hydroxyl making α-D-glycopyranoside units is exposed to the environment (for example, solvent) (see such as Fig. 1) of surrounding.Term " inertia cyclodextrin " refers to containing having basic form C6H12O6α-D-glycopyranoside units and do not have any other chemistry to take The cyclodextrin of the glucose structure in generation (alpha-cyclodextrin for example, with 6 glucose monomers, the β with 7 glucose monomers- Cyclodextrin, and there is the gamma-cyclodextrin of 8 glucose monomers).Term " phase in cyclodextrin " refers to be included in (that is, being encapsulated in) The relatively fewer hydrophilic region of the ring topology cloth intra-office of cyclodextrin structure.Term " cyclodextrin foreign minister " refer to not by cyclodextrin The region of the ring topology layout cincture of structure simultaneously can include, for example, the aqueous environments that exists during system vivo medicine-feeding or Refer to phase in the structure of oneself's encapsulating selective ATP generation inhibitor/cyclodextrin complexes.Cyclodextrin is used to dissolve hydrophobic components (see, for example, Albers and Muller (1995)Crit. Rev. Therap. Drug Carrier Syst. 12:311- 337;Zhang and Ma (2013)Adv. Drug Delivery Rev.65:1215-1233;Laza-Knoerr etc. (2010) J. Drug Targ.18:645-656;Challa etc. (2005)AAPS PharmSci. Tech. 6:E329- 357;Uekama etc. (1998)Chem. Rev. 98:2045-2076; Szejtli (1998) Chem. Rev. 98: 1743-1754;Stella and He (2008)Toxicol. Pathol.36:30-42;Rajewski and Stella (1996) J. Pharm. Sci. 85:1142-1169; Thompson (1997) Crit. Rev. Therap. Drug Carrier Sys.14:1-104；With Irie and Uekama (1997)J. Pharm. Sci. 86:147-162).It is positioned at ring In dextrin, any material in phase is considered as " encapsulating ".
As used herein, the cyclodextrin according to the present invention is useful, as long as cyclodextrin can be encapsulated selective ATP and produce Inhibitor.In some embodiments, cyclodextrin also carries ionogenic (for example, alkalescent and/or faintly acid) sense Group is to improve the stability that selective ATP produces inhibitor.So-called protective seletion ATP produces the stability of inhibitor, it is intended that Selective ATP produces inhibitor/cyclodextrin complexes, and to make selective ATP produce inhibitor molecules more stable, as by with discord Photostability that the stability of inhibitor molecules that produces the compound selective ATP of cyclodextrin compares, the stability of shelf life, heat Stability, anti-intramolecular cyclization stability, shown in acid-hydrolyzed stability, the anti-general fall stability of solution etc..
In order to encapsulate required therapeutic agent, cyclodextrin can add with effective, height-concentration according to the feature of required therapeutic agent It is loaded in parameter therein to select and/or be modified by sulphation.For example, it is preferable to cyclodextrin itself has the high dissolving in water Property, to promote therapeutic agent, the loading of such as 3-halogen acetone acid.In some embodiments, the water solubility of cyclodextrin is at least 10 mg/mL, 20 mg/mL, 30 mg/mL, 40 mg/mL, 50 mg/mL, 60 mg/mL, 70 mg/mL, 80 mg/ ML, 90 mg/mL, 100 mg/mL or higher.The water miscible method realizing such raising is well known in the art.
In some embodiments, big association constant relevant with therapeutic agent is preferred and can be by based on controlling Treat the number of glucose unit in the size Selection cyclodextrin of agent and obtain (see, for example, Albers and Muller (1995)Crit. Rev. Therap. Drug Carrier Syst.12:311-337;Stella and He (2008)Toxicol. Pathol.36:30-42；With Rajewski and Stella (1996)J. Pharm. Sci.85:1142-1169).As a result, In the presence of cyclodextrin, the dissolubility (name solubility) of therapeutic agent is available further to be improved.For example, cyclodextrin with control Treating the association constant of agent can be the 100th, the 200th, the 300th, the 400th, the 500th, the 600th, the 700th, the 800th, the 900th, 1,000, or higher.
By being formed with cyclodextrin hydroxy groups (for example, those hydroxyls of the ridge up and down at inertia cyclodextrin anchor ring for the lining) reaction Derivative can easily be prepared and be provided a kind of method of physicochemical properties modifying parent (inertia) cyclodextrin.One In a little embodiments, inertia cyclodextrin molecular or the physics and chemistry not producing the compound cyclodextrin molecular of inhibitor with selective ATP Matter is different from the character producing the compound cyclodextrin molecular of inhibitor with selective ATP.Therefore, compound with cyclodextrin selectivity ATP produces inhibitor molecules can be by observing dissolubility, chemical reactivity, UV/VIS absorbance, medicine retention, chemical stability Deng change and identify.For example, modification hydroxyl is had determined that herein, for example those hydroxyls away from phase in cyclodextrin, available Ionogenic chemical group substitutes to promote therapeutic agent, the loading in modification cyclodextrin of such as slightly solubility or hydrophobic drug and It is stable.In one embodiment, there is at least one modification cyclodextrin with the substituted hydroxyl of ionogenic chemical group The charged moieties under the conditions of some solvent (for example, pH) will be caused.Term " charged cyclodextrin " refers to have one or more Its hydroxyl is by charged moieties and takes charged partially substituted cyclodextrin.Such part itself can be charged group or It can comprise with one or more charged moieties substituted organic moiety (for example, C1-C6Alkyl or C1-C6Alkyl ether moieties).
In one embodiment, " ionogenic " or " charged " part is weak ionogenic.Weak ionogenic part It is that those are or alkalescent or weakly acidic part.Alkalescent functional group (X) has according to CH3-X about 6.0-9.0, Between 6.5-8.5,7.0-8.0,7.5-8.0, and the pK being included between any scopea.Similarly, weakly acidic functional group (Y) Have according to CH3-Y about between 3.0-7.0,4.0-6.5,4.5-6.5,5.0-6.0,5.0-5.5, and be included in any model Log dissociation constant (pK between enclosinga).PKa parameter is measuring known to the acid/base character of a kind of material, the side that pKa measures Method is traditional and conventional in this area.For example, the pKa value of many weak acid by list in chemical and pharmacological reference book In.Seeing, for example, the IUPAC handbook (IUPAC Handbook of Pharmaceutical Salts) of pharmaceutical salts, by P. H. Stahl and C. G Wermuth, Wiley-VCH, 2002 edit；CRC handbook chemically and physically, the 82nd edition, by D. R. Lide edits, CRC Press, Florida, 2001, p. 8-44 to 8-56.Because band has more than an ionogenic base The cyclodextrin of group has second that each personal subscript represents and the pKa of group subsequently.
Representational anionicsite includes, and without any restrictions, succinyl group, carboxylate radical, carboxymethyl, sulfonyl, phosphorus Acid group, sulfoalkyl ether, sulfate radical, carbonate, thio-carbonate, thio-carbonate, phosphate radical, phosphonate radical, sulfonate radical, nitric acid Root and boronic groups.
Representational cationic moiety includes, but not limited to amino, guanidine and quaternary ammonium group.
In another embodiment, the cyclodextrin of modification is " polyanion " or " polycation ".Polyanion is tool Have more than one, cause net anion electric charge, the modification cyclodextrin of electronegative group more than two units.Poly-sun from Son be have more than one, cause net cationic charge, the modification cyclodextrin of positively charged group more than two units.
In another embodiment, the cyclodextrin of modification is " amphiphile that can be charged ".So-called " can be charged " mean Amphiphile has the pK in the range of pH 4-pH 8 or 8.5.Therefore amphiphile that can be charged can be a kind of weak acid or weak base. So-called " both sexes " refer herein to one and have existing anionic nature, have again the repairing of ionogen of cationic property Circlets dextrin, wherein: 1) at least one of cation and anionic amphiphilic thing, and optionally both can be charged, have to The charged group of a few pK having between 4 and 8 to 8.5,2) cationic charge is preferably at pH 4, and 3) anion Electric charge is preferably at pH 8-8.5.
In some embodiments, " ionogenic " or " charged " cyclodextrin is as an entirety, no matter polyion, two Property molecule, or other, be all weak ionogenic (that is, to have at about 4.0-8.5,4.5-8.0,5.0-7.5,5.5- 7.0th, between 6.0-6.5, and the pKa of any scope being included in1)。
Cyclodextrin any one, some or all α-D-glycopyranoside units any one, some or all hydroxyls Base as described herein can be modified to ionogenic chemical group.Due to each cyclodextrin hydroxy groups in terms of chemical reactivity not With the reaction with modification part can produce the amorphous mixture of a kind of position and optical isomer.Alternatively, some chemistry α-D-the glycopyranoside units that can allow pre--modification reacts to form uniform product.
The total replacement occurring is by the term description of referred to as substitution value.For example, have 6-second diaminourea-β that substitution value is 7- The distribution of the isomers by 6-second diamino group-beta-cyclodextrin is formed by cyclodextrin, and wherein each 6-second diamino group-beta-cyclodextrin divides The average of the second diaminourea of son is 7.Substitution value can pass through mass spectrum or NMR spectrum determines.In theory, α-ring is stuck with paste Essence, maximum substitution value is 18, is 21 for beta-schardinger dextrin, and is 24 for gamma-cyclodextrin, but, there is the substituent of hydroxyl There is the possibility to other hydroxy alkylated in itself.The 1st, the 2nd, the 3rd, the 4th, the 5th, the 6th, the 7th, the 8th, the 9th, the 10th, the 11st, the 12nd, the 13rd, substitution value can be 14th, the 15th, the 16th, the 17th, the 18th, the 19th, the 20th, the 21st, the 22nd, the 23rd, 24, or more and can include replacing completely.
The hydroxyl substituted spatial chemistry positioning that another parameter is given.In one embodiment, at least one face Replaced by ionogenic chemical group to the hydroxyl away from cyclodextrin inner.For example, at least one α-D-glycopyranoside units C2, C3, C6, C2 and C3 of C2-C3-C6 hydroxyl, C2 and C6, C3 and C6, and all 3 taken by ionogenic chemical group Generation.Such carbon location is well known in the art.For example, the CH2OH shown in FIG of each α-D-glycopyranoside units Part represents C6 carbon.Any such combination of hydroxyl can similarly with at least the 2 of modification cyclodextrin, the 3rd, the 4th, the 5th, the 6th, the 7th, the 8th, the 9th, 10th, 11, until whole α-D-glycopyranoside units combines and combines with any substitution value described herein.
It is also acceptable for merging one or more cyclodextrin described herein.
C. ATP produce selective depressant and related compound
Some embodiments of the present invention relate to encapsulating in cyclodextrin for the selective depressant of ATP generation." ATP produces term Raw selective depressant " refers to that (for example, the enzymatic processes being generated ATP by interference suppresses the antimetabolic agent of ATP production The acid of GAPDH inhibitor such as 3-halogen acetone is as 3-bromo acetone acid).In some embodiments, the Selective depression that ATP produces Agent is " anti-tumor alkylating agent ", and it refers to the medicine causing hydrogen to be substituted by alkyl for treatment of cancer.As the term is employed herein " alkyl " refers to C1-20(including 1 and 20 end values) linear (that is, straight chain "), side chain, or ring-type, saturated or at least in part and Completely undersaturated (that is, thiazolinyl and alkynyl) alkyl under certain situation, its be derived between containing 1 and 20 carbon atom, pass through Remove the hydrocarbon part of single hydrogen atom.Representational alkyl includes, but not limited to methyl, ethyl, n-propyl, isopropyl, n- Butyl, isobutyl group, sec-butyl, tert-butyl, n-amyl, sec-amyl group, iso-amyl group, neopentyl, n-hexyl, sec-hexyl, N-heptyl, n-octyl group, n-decyl, n-undecyl, dodecyl etc., vinyl, acrylic, cyclobutenyl, pentenyl, oneself Thiazolinyl, octenyl, butadienyl, propinyl, butynyl, pentynyl, hexin base, heptynyl, and allene base (allenyl) base Group." branch " refers to alkyl, wherein low alkyl group, such as methyl, ethyl or propyl group, is connected to linear alkyl chain." lower alkyl Base " refers to have about 8 carbon atom (that is, C of 1-1-8Alkyl), for example, the 1st, the alkyl of the 2nd, the 3rd, the 4th, the 5th, the 6th, 7 or 8 carbon atoms." senior Alkyl " refers to have about 20 carbon atoms of about 10-, for example, and the 10th, the 11st, the 12nd, the 13rd, the 14th, the 15th, the 16th, the 17th, the 18th, 19 or 20 carbon atoms Alkyl.In certain embodiments, " alkyl " particularly relates to C1-8Straight chained alkyl.In other embodiments, " alkyl " particularly Refer to C1-8Branched alkyl.
Alkyl group is optionally substituted with one or more alkyl groups base and replaces (" substituted alkyl "), and described substituent can To be same or different.Term " alkyl substituent " include but is not limited to alkyl, substituted alkyl, halo, arylamino, Acyl group, hydroxyl, aryloxy group, alkoxyl, alkyl sulfenyl, artyl sulfo, aralkyl oxy, aromatic alkyl sulfurio, carboxyl, alkoxyl carbonyl Base, oxo, and cycloalkyl.Optionally insert one or more oxygen, sulphur or substituted or unsubstituted nitrogen along alkyl chain former Son, wherein nitrogen substituent is hydrogen, low alkyl group (being also referred to as " alkylaminoalkyl group " at this), or aryl.Therefore, as used herein , term " substituted alkyl " includes alkyl group as defined herein, wherein one or more atom of alkyl or functional group With another atom or functional group, including for example, alkyl, substituted alkyl, halogen, aryl, substituted aryl, alkoxyl, hydroxyl Base, nitro, amino, alkyl amino, dialkyl amido, sulfuric ester and sulfydryl substitute.
In one embodiment, the selective depressant that ATP produces usually is expressed from the next:
Wherein X represents halide, sulphonic acid ester, carboxylate, alkoxide or amine oxide.In certain embodiments, X is to be selected from Following halide: fluoride, bromide, chloride and iodide.In one embodiment, inhibitor is 3-halogen acetone Acid.In some other embodiment, 3-halogen acetone acid is selected from: 3-fluoropyruvate, the acid of 3-acetone dichloride, 3-bromo acetone Acid and 3-iodo pyruvic acid.In one embodiment, 3-halogen acetone acid is 3-bromo acetone acid.In other embodiments In, X is sulphonic acid ester and is selected from: triflate, methanesulfonates and tosylate.In a still further embodiment, X is Amine oxide such as dimethylamine oxide.In certain embodiments, R1Expression OR, H, N (R ")2, C1-C6 alkyl, C6-C12 virtue The miscellaneous alkyl of base, C1-C6, or C6-C12 heteroaryl.Independently, in other embodiments, R " represents H, C1-C6 alkyl, or C6- C12 aryl.Independently, in other embodiments also having, R represents H, alkali metal, C1-C6 alkyl, C6-C12 aryl or C (O)R’；With R ' represents H, C1-C20 alkyl or C6-C12 aryl.
In a preferred embodiment, the present invention also provides the inhibitor that ATP expressed by the following formula produces:
X-CH2-CO-COOH,
Wherein X represents halide, sulphonic acid ester, carboxylate, alkoxide or amine oxide.In certain embodiments, X is halogenation Thing is simultaneously selected from: fluoride, bromide, chloride and iodide.In one embodiment, inhibitor is 3-halogen acetone Acid.In certain embodiments, 3-halogen acetone acid is selected from: 3-fluoropyruvate, 3-acetone dichloride acid, 3-bromo acetone acid and 3-iodo pyruvic acid.In one embodiment, 3-halogen acetone acid is 3-bromo acetone acid.In other embodiments, X is Selected from following sulphonic acid ester: triflate, methanesulfonates and tosylate.In a still further embodiment, X is amine oxygen Compound such as dimethylamine oxide.
It is used as other analogs, derivative, prodrug, metabolin and the salt thereof of 3-bromo acetone acid, as long as these are changed Compound or composition have the antitumaous effect that statistics is similar to 3-bromo acetone acid.When use 3-bromo acetone acid mentioned above During treatment, it should be appreciated that also can be with the analog of 3-bromo acetone acid, derivative, prodrug, metabolin and salt in where applicable treatment Carry out.
D. cyclodextrin/ATP inhibitor combination
The present invention provides the selective depressant in the cyclodextrin comprising to be encapsulated in inertia and/or modification, above-mentioned ATP produces Pharmaceutical composition.Such compound is referred to herein as cyclodextrin/ATP inhibitor combination.The Selective depression that ATP produces Agent can be 1:1 with the ratio of cyclodextrin, so that an inhibitor molecules forms compound with a cyclodextrin molecular.Or, Described ratio can be 2:1,3:1,4:1,5:1, or more.
On the one hand, the present invention provides pharmaceutically acceptable composition, and it comprises pharmaceutically can connect with one or more The carrier (additive) being subject to and/or diluent is formulated together, the said one of therapeutically effective amount or multiple such ring are stuck with paste Essence/ATP inhibitor.On the other hand, described composition can former state or mix with pharmaceutically acceptable carrier and give and also may be used With other anti-cancer therapy, such as chemotherapeutics, scavenger compounds, radiotherapy, biotherapy etc. is combined and is given.Therefore, combine Therapy includes sequentially, simultaneously and separately, or jointly gives described composition, wherein when giving compound subsequently, for the first time The therapeutic action being administered not yet is wholly absent.
As detailed below, in order to give with solid or liquid form, the pharmaceutical composition of the present invention can be special Do not prepare, including those are suitable for the form that following approach gives: (1) is orally administered to, for example, oral liquid medicine (drenches) (aqueous or non-aqueous solution or suspension)/tablet, for example, those are for oral cavity, sublingual and whole body suction Receipts, bolus, powder, granule, the paste being applied to tongue；(2) parenteral give, for example, through subcutaneous, intramuscular, intravenous Or endocranium injection conduct, for example, sterile solution or suspension, or sustained release preparation；(3) topical application, for example, as one Creme, ointment, or control-released plaster or the spray being applied to skin；(4) in vagina or rectum, for example, as a kind of cloudy Road suppository, creme or foaming agent；(5) sublingual administration；(6) intraocular；(7) percutaneous；Or (8) intranasal.
As raised above, some embodiment of selective ATP inhibitor or cyclodextrin/ATP inhibitor combination can Containing basic functionality, such as amino or alkyl amino, and therefore, it is possible to pharmaceutically-acceptable acid formed pharmaceutically-can The salt accepting.These salt can give medium or prepared by dosage form manufacturing process situ, or by making the basis of its free alkali form The purifying compound of invention react with suitable organic or inorganic acid respectively, and separation is thusly-formed during purifying subsequently Salt.Representational salt includes hydrobromate, hydrochloride, sulfate, disulfate, phosphate, nitrate, acetate, valerate, Oleate, palmitate, stearate, laruate, benzoate, lactate, phosphate, toluene fulfonate, citrate, Maleate, fumarate, succinate, tartrate, naphthoate, mesylate, gluceptate, Lactobionate, and ten Dialkyl group sulfonate etc. (see, for example, Berge etc. (1977) " pharmaceutical salts (Pharmaceutical Salts) ",J. Pharm. Sci. 66:1-19)。
The pharmaceutically acceptable salt of this compound includes for example, from the compound of non-toxic organic or inorganic acid Conventional non-toxic salts or quaternary ammonium salt.For example, such conventional non-toxic salts include being derived from inorganic acid example hydrochloric acid, hydrobromic acid, sulfuric acid, Those salt of sulfamic acid, phosphoric acid, nitric acid etc.；And from organic acid such as acetic acid, propionic acid, butanedioic acid, glycolic, stearic acid, breast Acid, malic acid, tartaric acid, citric acid, ascorbic acid, palmitic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzene first Acid, salicylic acid, p-aminobenzene sulfonic acid, Aspirin, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethionic acid, hydroxyl second The salt of the preparation such as sulfonic acid (isothionic).
In other situations, the selective ATP inhibitor of the present invention or cyclodextrin/ATP inhibitor combination can contain one Individual or multiple acidic functionalities, therefore, it is possible to form pharmaceutically-acceptable salt with pharmaceutically-acceptable alkali.These salt can Equally giving medium or prepared by dosage form manufacturing process situ, or by make the purifying compound of its free acid form respectively with Suitable alkali, the hydroxide of for example pharmaceutically-acceptable metal cation, carbonate or bicarbonate, and ammonia, or and medicine On-acceptable organic primary amine, secondary amine or reactive tertiary amine.Representational alkali metal or alkali salt include lithium, sodium, potassium, Calcium, magnesium, and aluminium salt etc..To formed the useful representative organic amine of base addition salts include ethamine, diethylamine, ethylenediamine, monoethanolamine, Diethanol amine, piperazine etc. (see, for example, Berge etc., ibid).
Wetting agent, emulsifying agent and lubricant, such as lauryl sodium sulfate and magnesium stearate, and colouring agent, releasing agent, Coating agent, sweetener, flavouring and fumet, preservative and antioxidant also are present in described composition.
The example of pharmaceutically-acceptable antioxidant includes: (1) water soluble antioxidant, such as ascorbic acid, salt Acid cysteine, niter cake, sodium metabisulfite, sodium sulfite etc.；(2) oil-soluble antioxidants, such as Vitamin C Acid palmitate, Butylated Hydroxytoluene (BHA), fourth hydroxyl return ether (BHT), lecithin, propylgallate, alpha-tocopherol etc.；(3) Metal-chelator, such as citric acid, ethylenediamine tetra-acetic acid (EDTA), sorbierite, tartaric acid, phosphoric acid etc..
Cyclodextrin/ATP inhibitor combination preparation includes being suitable for being administered orally, intranasal, locally (include under cheek and sublingual), straight Intestines, vagina and/or those giving parenteral.Preparation advantageously can exist with unit dosage forms and can be by known to pharmaceutical field Prepared by any method.Can with carrier material be combined to produce the amount of the active component of single formulation by according to host to be treated and Specific mode of administration and change.Can be combined to produce the amount of the active component of single formulation by usually generation with carrier material The amount of the compound of therapeutic action.
In certain embodiments, the preparation of cyclodextrin/ATP inhibitor combination can comprise other carriers to allow more Stable, different internal release characteristics, target specific site, more effective transmission compound or will be allowed to experimenter or tested Any other required characteristic of the target in person's body, such as, but not limited to, liposome, microballoon, nanosphere, nano particle, liquid Bubble, micelle forming agent, for example, cholic acid, and polymer support, for example, polyester and polyanhydride.In certain embodiments, aforementioned system Agent gives the oral bioavailability of the compound of the present invention.
The liquid dose formulations of cyclodextrin/ATP inhibitor combination includes pharmaceutically acceptable emulsion, microemulsion, solution Agent, supensoid agent, syrup and elixir.Except active component, liquid dosage form can contain the inert diluent being generally used for this area, For example, water or other solvents, solubilizer and emulsifying agent, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzylalcohol, benzene first Acid benzyl ester, propane diols, 1,3-BDO, oil (particularly, cottonseed oil, peanut oil, corn oil, embryo oil, olive oil, castor oil and Sesame oil), glycerine, tetrahydrofurfuryl carbinol, the fatty acid ester of polyethylene glycol and anhydro sorbitol, and mixture.
Except inert diluent, Orally administered composition may also comprise adjuvant such as wetting agent, emulsifying agent and suspending agent, sweet taste Agent, flavouring, colouring agent, spices and preservative.
Supensoid agent, except active ingredient beyond the region of objective existence, also can containing suspending agent for example, ethoxylated isostearyl alcohols, polyoxyethylene Sorbierite and Isosorbide Dinitrate, microcrystalline cellulose, inclined aluminium hydroxide, bentonite, aga agar and bassora gum, and mixing Thing.
The preparation being suitable for being orally administered to can be rendered as capsule, cachet, pill, tablet, the lozenge (base of use seasoning Matter, usually sucrose and Arabic gum or bassora gum), powder, granule, or as a kind of in aqueous or non-waterborne liquid Solution or supensoid agent, or as a kind of oil-in-water or water-in-oil liquid emulsion, or as a kind of elixir or syrup, or make For lozenge (using inactive alkali, such as gelatin and glycerine, or sucrose and Arabic gum) and/or as forms such as gargles, each Active component containing scheduled volume.The cyclodextrin of the present invention/ATP inhibitor combination also can as bolus, electuary or Paste gives.
In solid dosage forms (for example, capsule, tablet, pill, dragee, powder, granule etc.), active component and one Individual or multiple pharmaceutically-acceptable carrier, such as sodium citrate or Dicalcium Phosphate, and/or any following material mixing: (1) Filler or extender, such as starch, lactose, sucrose, glucose, mannitol, and/or silicic acid；(2) adhesive, for example, carboxylic Methylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and/or acacia；(3) NMF, such as glycerine；(4) Disintegrant, such as aga agar, calcium carbonate, potato or tapioca, alginic acid, some silicate, and sodium carbonate；(5) molten The sluggish agent of liquid, such as paraffin；(6) accelerator, such as quaternary ammonium compound are absorbed；(7) wetting agent, for example, hexadecanol, a tristearin Acid glyceride, and non-ionic surface active agent；(8) absorbent, such as kaolin and bentonite；(9) lubricant, such as talcum Powder, calcium stearate, magnesium stearate, solid polyethylene glycol, lauryl sodium sulfate, and mixture；(10) colouring agent.? In the case of capsule, tablet and pill, composition also can comprise buffer.The solid constituent of similar type also is used as soft Filler in firmly-shell gelatine capsule agent, uses such excipient such as lactose or toffee, and high molecular weight polyethylene glycol Deng.
Tablet is optionally prepared by compacting or molding by one or more auxiliary elements.The tablet of compacting can use viscous Mixture (for example, gelatin or hydroxypropyl methyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, hydroxyl second Acid-starch sodium or Ac-Di-Sol), prepared by surface-active agent or dispersant.Molded tablet can be by closing The mixture of the powdered compounds that suitable machine mold pressing is moistened by inert liquid diluent prepares.
Tablet, and other solid dosage forms, such as dragee, capsule, pill and granule, can optionally be scored or with bag Prepared by dress material and shell, such as enteric coating and other coating material known in pharmaceutical-formulating art.They also can be formulated to There is provided active component slowly or control release, wherein the ratio with change uses such as hydroxypropyl methyl cellulose (to provide The release characteristics needing), other polymeric matrixs, liposome and/or microballoon.Composition also can be formulated for quickly discharging, example Such as freeze-drying.They can pass through, and for example, is filtered by bacterial filter, or by combining in aseptic solid composite form , the bactericidal agent dissolving in sterilized water, or some other in the aseptic injectable medium sterilizing using before use.These Composition also optionally contain opacifier and can having make they only (or preferably) in some part GI, optionally Ground discharges the composition of active component in a delayed fashion.The example of spendable embedding component includes polymer material and wax.As When fruit is suitable for, active component also can be rendered as the form of the microcyst containing one or more above-mentioned excipient.
The preparation giving for rectum or vagina can exist as suppository, and it can be by the one or more present invention's of mixing Compound and one or more suitable non-irritating excipients or carrier (include, for example, cocoa butter, polyethylene glycol, suppository wax Or salicylate) prepare, and it is solid at room temperature, but be liquid under body temperature, therefore, will be in rectum or vaginal canal Melt and release of active compounds.
It is suitable for the preparation that vagina gives also to include containing as known in the art for the vaginal plug of suitable examples of such carriers Agent, vagina plug (tampons), creme, gel, paste, foaming agent or spray formu.
Formulation for local or the cyclodextrin/ATP inhibitor combination percutaneously giving the present invention includes powder, spraying Agent, ointment, paste, creme, lotion, gel, solution, patch and inhalant.Reactive compound can be aseptically With pharmaceutically-acceptable carrier, and with any preservative, buffer solution, or may need propellant mixing.
Except the active ingredient beyond the region of objective existence of the present invention, ointment, paste, creme and gel also can contain excipient, for example Animal and plant fat, oil, wax, paraffin, starch, bassora gum, cellulose derivative, polyethylene glycol, silicone, bentonite, silicic acid, Talcum powder and zinc oxide, or its mixture.
Powder and spray can be containing excipient such as lactose, talcum powder, silicic acid, aluminium hydroxide, calcium silicates and Silons End, or the mixture of these materials.Spray can additionally contain conventional propellant, for example chlorofluoro hydrocarbon and volatile Unsubstituted hydrocarbon, such as butane and propane.
Transdermal skin patches has the additional advantage providing control to be transferred to health.Such formulation by dissolving or can be disperseed Compound prepares in suitable medium.Absorption enhancer also can be used to increase the flux that compound crosses skin.Such The speed of flux can be controlled by providing rate controlling membranes or be scattered in described compound in polymer substrate or gel.
Eye-drops preparations, Eye ointments, powder agent, solution etc. are also included in the scope of the present invention.
Be applicable to the parenteral pharmaceutical composition giving can comprise that sterile isotonic is aqueous or non-aqueous solution agent, dispersant, Supensoid agent or emulsion, or aseptic powdery agent, it can reconstitute for sterile injectable solution agent or dispersion liquid before use, and it can Containing sugar, alcohol, antioxidant, buffer solution, bacteriostatic agent, make the isotonic solute of the blood of preparation and intended recipient or suspending agent or Thickener.
The example of the suitable aqueous and non-aqueous carrier that can be used for the pharmaceutical composition of the present invention includes water, ethanol, polynary Alcohol (such as glycerine, propane diols, polyethylene glycol etc.), and suitable mixture, vegetable oil, such as olive oil, and injectable Organic ester, such as ethyl oleate.Suitable mobility can for example, by using coating material, such as lecithin, at dispersion liquid In the case of by maintaining required granularity, and maintained by using surfactant.
In certain embodiments, aforementioned pharmaceutical compositions can according to method and composition provided herein with this area Other pharmaceutically active compounds (" second activating agent ") known combine.Second activating agent can be big molecule (for example, albumen Matter) or little molecule (for example, inorganic, the organic metal of synthesis, or organic molecule).In one embodiment, second activity Agent helps to treat cancer independently or synergistically.
For example, chemotherapeutics is anti-cancer agent.Term chemotherapeutics includes, but not limited to platinum-base medicine, for example carboplatin and suitable Platinum；Mustargen alkylating agent；Nitrourea alkylating agent, such as BCNU (BCNU) and other alkylating agents；Antimetabolic agent, such as ammonia first Petrin；Purine analogue antimetabolic agent；Pyrimidine analogue antimetabolic agent, such as floxuridine (5-FU) and gemcitabine；Swash Element antitumor agent, such as Goserelin, leuproside, and TAM；Natural antitumor agent, such as taxanes are (for example, many Xi Tasai and taxol), Aldesleukin, proleulzin, Etoposide (VP-16), interferon-' alpha ', and Tretinoin (ATRA)；Anti- The natural antitumor agent of raw element, such as bleomycin, actinomycin D, daunorubicin, Doxorubicin, and mitomycin；Raw with Changchun Alkaloids natural antitumor agent, such as vincaleukoblastinum and vincristine.
Additionally, following medicine also can be applied in combination with antitumor agent, even if not considering antitumor agent itself: actinomycin D； Daunorubicin HCl；Docetaxel；Doxorubicin HCl；Epoetin (epoetin) α；Etoposide (VP-16)；GCV Sodium；Gentamicin sulphate；Interferon-' alpha '；Acetic acid leuproside；Pethidine (meperidine) HCl；Methadone (methadone) HCl；Ranitidine HCl；Vinblastine sulfate；With Zidovudine (AZT).For example, floxuridine recently with adrenaline and ox glue Former protein combination is prepared to form a kind of particularly effective combination.
Further, following amino acid, peptide, polypeptide, protein, polysaccharide, and the list of other big molecules may be used without: Interleukin-11-18, including include mutant and analog；Interferon or cell factor, such as interferon-' alpha ', β and γ；Hormone, example Such as luteinizing hormone-releasing hormone (LRH) (LHRH) and analog and, gonadotropin-releasing hormone (GnRH)；Growth factor, for example, convert Growth factor-beta (TGF-β), fibroblast growth factor (FGF), nerve growth factor (NGF), somatotropin releasing factor (GHRF), EGF (EGF), fibroblastic growth factor autofactor 1 (FGFHF), HGF , and insulin-like growth factor (IGF) (HGF)；Tumor necrosis factor-alpha & β (TNF-α & β)；Invasion and attack inhibiting factor-2 (IIF-2)；Bone morphogenetic protein 1-7 (BMP 1-7)；Growth hormone release inhibiting hormone；Thymosin extrasin-α-1；Gamma globulin；Superoxides discrimination Change enzyme (SOD)；Complement factor；Anti-angiogenesis；Antigen-like material；And prodrug.
Include, but are not limited to alkylating agent such as plug with the chemotherapeutics that therapeutic combination described herein and method are used together to replace Group and endoxan；Alkylsulfonate such as busulfan, Improsulfan (improsulfan) and piposulfan (piposulfan)； Aziridine such as Benzodepa (benzodopa), card ripple quinone, Meturedepa (meturedopa), and uredepa (uredopa)；Triethylenemelamine class (ethylenimines) and methylmelamine class (methylamelamines) include pregnancy honey Amine, tretamine, APO, triethylenethiophosphoramide and trimethylolmelamine；Acetogenin class (acetogenins) (particularly bullatacin and bullatacin ketone)；Camptothecine (includes the analog Hycamtin of synthesis)；Bryostatin (bryostatin)；callystatin；CC-1065 (includes that its Adozelesin, Carzelesin are similar with Bizelesin synthesis Thing)；Hidden algae element class (cryptophycins) (particularly hidden algae element 1 and hidden algae element 8)；Dolastatin (dolastatin)； Times carcinomycin (duocarmycin) (includes the analog of synthesis, KW-2189 and CB1-TM1)；Soft coral alcohol (eleutherobin)；Water ghost any of several broadleaf plants alkali (pancratistatin)；Crawl a coral alcohol (sarcodictyin)；Sponge inhibin (spongistatin)；Nitrogen mustards such as Chlorambucil, Chlornaphazine (chlornaphazine), cholophosphamide, female Mo Siting, ifosfamide, mustargen, mustron, L-PAM, novoembichin (novembichin), phenesterin, bold and vigorous Buddhist nun Mo Siting, Trofosfamide, uracil mastard；Nitrosoureas such as BCNU, chlorozotocin (chlorozotocin), Fu Mosi Spit of fland (fotemustine), lomustine (lomustine), Nimustine (nimustine) and Ranimustine (ranimustine)；Antibiotic such as enediyne (enediyne) antibiotic (for example, calicheamycin (calicheamicin), special It not calicheamycin γ lI and calicheamycin ω l1；Enediyne anthracycline antibiotic, including enediyne anthracene nucleus A；Diphosphonate, example Such as clodronate；Ai Sipeila mycin (esperamicin)；And neoearcinostain chromophore and related chromoprotein enediyne Antibiotic chromophore, aclacinomycin (aclacinomysins), D actinomycin D, anthramycin (authramycin), diazonium silk Propylhomoserin, bleomycin, D actinomycin D c, carabicin, carminomycin (caminomycin), cardinophyllin (carzinophilin), chromomycin (chromomycin), actinomycin D, daunorubicin, Detorubicin (detorubicin), 6-diaza-5-oxn-l-norieucin, Doxorubicin (include morpholino-Doxorubicin, Cyanomorpholino-Doxorubicin, 2-pyrrolin subbase-Doxorubicin and deoxydoxorubicin), epirubicin, esorubicin (esorubicin), idarubicin (idarubicin) ripple mycin (marcellomycin), mitomycin such as mitomycin C, mycophenolic acid, nogalamycin, are sent out (nogalamycin), olivomycin (olivomycins), Peplomycin, porfiromycin (potfiromycin), puromycin (puromycin), triferricdoxorubicin (quelamycin), rodorubicin (rodorubicin), streptonigrin, streptozotocin (streptozocin), tubercidin, ubenimex (ubenime), Zinostatin, zorubicin；Anti-metabolin such as ammonia first Petrin and 5-FUD (5-FU)；Folacin such as denopterin (denopterin), methotrexate, pteropterin (pteropterin), Trimetrexate (trimetrexate)；Purine analogue such as fludarabine, Ismipur, imuran Amine (thiamiprine), thioguanine；Pyrimidine analogue such as ancitabine (ancitabine), azacitidine (azacitidine), 6-azauridine (6-azauridine), Carmofur (carmofur), cytarabine (cytarabine), double BrdU (dideoxyuridine), doxifluridine (doxifluridine), enocitabine, floxuridine (floxuridine)；Male sex hormone such as clausterone (calusterone), dromostanolone propionate (dromostanolone Propionate), epithioandrostanol (epitiostanol), Mepitiostane (mepitiostane), Testolactone (testolactone)； Anti-adrenal gland medicine (anti-adrenals) such as aminoglutethimide (aminoglutethimide), mitotane (mitotane), Qu Luo Take charge of smooth (trilostane)；Folic acid compensation such as folinic acid (frolinic acid)；Aceglatone (aceglatone)；Aldehyde Phosphamide glucosides (aldophosphamide glycoside)；Aminolevulinic acid (aminolevulinic acid)；Grace urine is phonetic Pyridine (eniluracil)；Amsacrine (amsacrine)；Beta cloth pungent (bwstrabucil), bisantrene (bisantrene)；Depend on Reach Qu Sha (edatraxate)；Defosfamide (defofamine)；Demecolcine (demecolcine)；Diaziquone (diaziquone)；Eflornithine (elfornithine)；Elliptinium acetate (elliptinium acetate)；Angstrom rich mould Element (epothilone)；Ethoglucid (etoglucid)；Gallium nitrate；Hydroxycarbamide；Lentinan (lentinan)；Lonidamine (lonidainine)；Maytansinoid class (maytansinoid) such as maytansine and ansamitocin (ansamitocins)； Mitoguazone (mitoguazone)；Mitoxantrone (mitoxantrone)；mopidanmol；Nitragin (nitraerine)； Pentostatin；Phenamet (phenamet);THP；Losoxantrone (losoxantrone)；Podophyllic acid (podophyllinic acid)；2-ethyl hydrazides；Procarbazine；PSK polysaccharide compound)；Tetrahydroform (razoxane)；Root nodule Rhzomorph (rhizoxin)；Sizofiran (sizofuran)；Spiral shell germanium (spirogermanium)；Tenuazonic acid (tenuazonic acid)；Triethyleneiminobenzoquinone (triaziquone)；2,2', 2''-tri-chloro triethylamine；Trichothecenes Compound (trichothecenes) (particularly T-2 toxin, myconomycin A (verracurin A), Roridine A (roridin A) and anguidin (anguidine))；Urethane (urethan)；Eldisine；Dacarbazine (dacarbazine)；Mannomustine (mannomustine)；Dibromannitol (mitobronitol)；Mitolactol (mitolactol)；Pipobroman (pipobroman)；gacytosine；Cytarabine (arabinoside) (" Ara-C ")； Endoxan；Phosphinothioylidynetrisaziridine；Taxanes, such as taxol, docetaxel；Chlorambucil；Gemcitabine；6-thioguanine；Mercapto Base purine；Methotrexate；Platinum coordination complex such as cis-platinum, oxaliplatin and carboplatin；Vincaleukoblastinum；Platinum；Etoposide (VP-16)； Ifosfamide；Mitoxantrone；Vincristine；Vinorelbine；Mitoxantrone hydrochloride；Teniposide；Edatrexate；Soft red mould Element；Aminopterin；Xeloda (xeloda)；Ibandronate；Irinotecan (for example, CPT-11)；Topoisomerase enzyme inhibitor RFS 2000；DFMO (DMFO)；Retinoid medicine such as vitamin A acid；Capecitabine；And any of the above medicine Pharmaceutically acceptable salt, acid or derivative.
In another embodiment, the composition of the present invention can comprise other bioactivators, including medicine Or prodrug, for example, other chemotherapeutics, scavenger compounds, antibiotic, anti-viral agent, anti-epiphyte pharmaceutical, anti-scorching medicine, blood vessel are received Contracting agent and anticoagulant, the antigen being used for cancer vaccine application or corresponding prodrug.
Exemplary scavenger compounds includes, but are not limited to the compound containing sulfydryl such as glutathione, thiocarbamide, and half Guang Propylhomoserin；Alcohol such as mannitol, substituted phenols；Quinones, substituted phenols, arylamine class and nitro compound.
Various forms of chemotherapeutics and/or other bioactivators can be used.These include, but not limited to for example without The forms such as the molecule of electric charge, molecular complex, salt, ether, ester, acid amides, which is BA.
E. the method preparing cyclodextrin/ATP inhibitor combination
Prepare the method for cyclodextrin/ATP inhibitor combination and preparation thereof and include making the compound of the present invention and carrier and optional One or more auxiliary elements mixing step.In general, the selection that described preparation is produced by making ATP as herein described Property is prepared closely mix all even with cyclodextrin of inhibitor.Usually, such compound can be by dropping therapeutic agent (for example, molten containing cyclodextrin of agitation and hybrid ring dextrin when (solution of selective depressant for example, producing containing ATP) Liquid) and obtain, or vice versa as the same。Many contributes to inhibitor and the mixed method of cyclodextrin merging is to it known in the art, example As but being not limited to, ultrasonically treated, eddy current, stirring, heating, co-precipitation, neutralization, slurrying, kneading, grinding etc..According to therapeutic agent Physical characteristic, it is possible to use and be dissolved in the material of solvent or solid matter as therapeutic agent.Solvent is had no particular limits, and And people can use, for example, the material identical with cyclodextrin foreign minister.The amount of the therapeutic agent mixing with cyclodextrin can be equimolar Amount or different ratios, this depends on combining required level.In some embodiments, the selective depressant that ATP produces Absolute magnitude can at the 0.001-10 mol equivalent of the amount relative to cyclodextrin, in 0.01-1 mol equivalent weight range, or comprise Any scope.Equally, heating-up temperature is had no particular limits.For example, 5 DEG C or higher, room temperature or higher (for example, 20 DEG C or Higher is also preferred), it is all acceptable.
Exist and remove any unwanted or unconjugated compound or composition, the treatment for example do not encapsulated by cyclodextrin Agent or not by the method known to liposomal encapsulated therapeutic agent cyclodextrin complexes.Representative example includes, but not limited to Analysis, centrifugation, and gel filtration.Dialysis can for example use dialysis membrane to carry out.As dialysis membrane, people can quote has molecule The film such as cellulose tube or Spectra/Por of amount cut-off.As for centrifugation, CENTRIFUGAL ACCELERATING preferably with 100,000 g or Higher, and more preferably with 300,000 g or higher is carried out.Gel filtration can, for example, by based on molecular weight use post such as Sephadex or Sepharose carries out fractionation and carries out.
In some cases, for extending medicine effect, (for example, slowing down) is changed from the drug absorption subcutaneously or intramuscularly injected It is desirable.This can be realized by using the liquid suspension with poor water miscible crystallization or amorphous materials. The absorptivity of medicine depends on its speed dissolved, and this can be depending on again crystalline size and crystal form successively.Alternatively, stomach Parenteral-the delayed absorption of medicament forms that gives can be by dissolving or suspended drug realize in oiliness medium.Implement at some In scheme, the selective ATP inhibitor combination of cyclodextrin-encapsulating described herein can be loaded in liposome.
Injectable reservoir type is by making this compound at biodegradable polymer such as polylactide-polyglycolide Middle formation micro encapsulating matrix prepares.According to the ratio of medicine and polymer, and the character of concrete polymer used, controlled pharmacy The speed of thing release.The example of other biodegradable polymer includes poly-(ortho esters) and poly-(acid anhydride).Reservoir is injectable Preparation also by being trapped in the liposome compatible with bodily tissue or microemulsion preparation by medicine.
F. treatment method
The present invention also provides prevention, delay, mitigates, and/or treatment cancer, including the new treatment method of cancerous tumour.One In individual embodiment, a kind for the treatment of method includes the cyclodextrin/choosing giving experimenter (for example, experimenter in need) effective dose Selecting property ATP produces inhibitor combination.Experimenter in need can include, for example, has diagnosed and has suffered from tumour, including front carcinous swollen Knurl, the experimenter of cancer, or the experimenter being treated, including to the previous unresponsive experimenter for the treatment of.
Biologically institute as needed for the term " effective dose " in the therapeutic agent of " therapeutically effective amount " has guided is required The amount of medicine.As will be recognized for those of ordinary skill in the art, the effective dose of medicine can be biological as required according to example Terminal, medicine to be transmitted, the composition of pharmaceutical composition, the factor such as target tissue or cell and change.More particularly, term " has Effect amount " refers to the amount that be enough to produce required effect, for example, reduces or alleviate disease, illness or the patient's condition, or one or more disease The seriousness of shape, duration, progress, or occur；The progress of prevention disease, illness or the patient's condition, causes disease, illness or the patient's condition Disappear；Prevent recurrence, the development of the symptom relevant with disease, illness or the patient's condition, occur or progress, or improve or improve another Plant prevention or the therapeutic action of therapy.
The method of the present invention can be used to treat any carcinous or front cancerous tumour.In certain embodiments, carcinous swollen Knurl has the glycolysis phenotype of height.For example, the glycolysis tumour of height can be located at selected from following tissue: brain, colon, uropoiesis Genitals, lung, kidney, prostate, pancreas, liver, oesophagus, stomach, hematopoietic tissue, mammary gland, thymus gland, testis, ovary, skin, marrow and/ Or uterine tissue.In some embodiments, the method and composition of the present invention can be used to treat any cancer.Can be by this The cancer of the method and composition treatment of invention includes, but not limited to from bladder, blood, bone, marrow, brain, mammary gland, colon, food The cancer in pipe, stomach and intestine, gums, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus is thin Born of the same parents.In addition, cancer can have following histological type especially, although it is not limited to these: tumour, malignant tumour；Cancer；Not The cancer of differentiation；Carcinoma gigantocellulare and carcinoma sarcomatodes；Small cell carcinoma；Papillary carcinoma；Squamous cell carcinoma；Lymphepithelioma；Substrate is thin Born of the same parents' cancer；Pilomatrix carcinoma (pilomatrix carcinoma)；Transitional cell carcinoma；Papillary transitional cell carcinoma；Gland cancer；Malignant Gastric is secreted Element knurl；Cholangiocarcinoma；Hepatocellular carcinoma；Compressibility hepatocellular carcinoma and cholangiocarcinoma；Girder gland cancer；Adenoid cystic carcinoma；In adenomatous polyp Gland cancer；Gland cancer, familial polyposis coli；Solid tumor；Malignant carcinoid tumours；Bronchiolo-alveolar adenocarcinoma；Papillary adenocarcinoma； Chromophobe cell tumor (chromophobe carcinoma)；Acidophil carcinoma；Oncocytic adenoma (oxyphilic adenocarcinoma)；Basophilic granulocyte cancer；Clear cell carcinoma；Granulocyte carcinoma；Follicular adenocarcinoma；Mamillary and filter blocking gland Cancer；Non-bladder sclerosing carcinoma (nonencapsulating sclerosing carcinoma)；Adrenocortical carcinoma；In uterus Film sample cancer；Skin appendage carcinoma (skin appendage carcinoma)；Apocrine adenocarcinoma (apocrine adenocarcinoma)；Carcinoma of sebaceous glands；Gland cancer of earwaxing (ceruminous adenocarcinoma)；Mucoepidermoid carcinoma；Capsule Gland cancer；Papillary cystic adenocarcinoma；Serous papillary cystadenocarcinoma；Mucus cystadenocarcinoma；Myxoadenocarcinoma；Signet ring cell cancer；Wellability Duct carcinoma；Cephaloma；Lobular carcinoma；Inflammatory carcinoma；Paget disease (paget's disease), breast cancer, acinar cell carcinoma；Glandular scale Cancer；Gland cancer w/ squamous metaplasia；Malignant thymoma；Malignant ovary mesenchymal neoplasm；Pernicious theca cell tumor；Pernicious granulocyte Knurl；With pernicious blastoma；Sertoli cell；Pernicious Leydig cell tumor (leydig cell tumor, malignant)；Pernicious lipid cell tumour；Pernicious Chromaffionoma；The outer Chromaffionoma of malignant galactophore；Pheochromocytoma；Blood Pipe ball sarcoma (glomangiosarcoma)；Malignant mela noma；Melanoma (the amelanotic of disposition without black melanoma)；Superficial spreading melanoma；Malignant mela noma in giant pigmented nevus；Epithelioid cell's melanoma；Dislike Property ble nevus (blue nevus, malignant)；Sarcoma；Fibrosarcoma；MFH；Myxosarcoma；Fat Sarcoma；Leiomyosarcoma；Rhabdomyosarcoma；Embryonal rhabdomyosarcoma；Alveolar rhabdomyosarcoma；Stromal sarcoma；Pernicious mixed Close knurl, mullerian mixed tumor (mullerian mixed tumor)；The nephroblastoma；Liver cancer (hepatoblastoma)；Cancer meat Knurl；Malignant stromal tumors；Malignant Brenner tumor (brenner tumor, malignant)；Pernicious phyllodes tumor；Synovial sarcoma；Dislike Property celiothelioma, dysgerminoma (dysgerminoma)；Embryonal carcinoma；Malignant teratoma；Malignant ovary adenoncus knurl；Choriocarcinoma； Pernicious mesonephroma (mesonephroma, malignant)；Angiosarcoma；MA；Kaposi's sarcoma；Pernicious Hemangiopericytoma；Lymphangioendothelial sarcoma；Osteosarcoma；Juxtacortical osteogenic sarcoma (juxtacortical osteosarcoma)；Soft Osteosarcoma；Pernicious chondrosarcoma；Mesenchimal chondrosarcoma；Giant cell tumor of bone；Ewing's sarcoma (ewing's sarcoma)；Pernicious odontogenic tumor；Ameloblastic odontosarcoma (ameloblastic odontosarcoma)；Pernicious one-tenth glaze Cytoma；Ameloblastic fibrosarcoma；Pernicious pinealoma；Chordoma；Glioblastoma；Ependymoma；Astrocytoma；Former Slurry property astrocytoma；Fibrillary astrocytoma；Astroblastoma；Glioblastoma；Oligodendroglioma；Become few Prominent spongiocytoma (oligodendroblastoma)；Original neural epiblast (primitive neuroectodermal)； Cerebellar sarcoma；Ganglioneuroblastoma；Neuroblastoma；Retinoblastoma；Olfactory neurogenic tumor；Pernicious meninx Knurl；Neurofibrosarcoma；Malignant schwannoma；Pernicious granulocyte knurl；Malignant lymphoma；Hodgkin's disease；He Jiejinshi lymph Knurl；Paragranuloma；Small lymphocyte malignant lymphoma；Diffusivity maxicell malignant lymphoma；Pernicious follicular lymphoma；Gill fungus Sample granuloma；Other non-He Jiejin lymphomas；Malignant histiocytosis；Huppert's disease；Mast cell meat Knurl；Immunoproliferative small intestinal disease；Leukaemia；Leukemic lymphoblastoid；Plasma cell leukemia；Erythroleukemia (erythroleukemia)；Lymphoma cell leukemia；Marrow series leukemia；Basophilic cell leukemia；Eosinophil Leukaemia；Monocytic leukemia；Mast cell leukemia；Megakaryoblast leukaemia；Medullary system sarcoma；The white blood with hair cell Sick.
Compositions described herein can be transmitted by any suitable method of administration, including be administered orally, intranasal, transmucosal, eye In, rectum, vagina, parenteral, including in intramuscular, subcutaneous, intramedullary injection, and sheath, direct ventricle is interior, intravenous, joint In, in breastbone, intrasynovial, in liver, in focus, encephalic, intraperitoneal, intranasal, or intraocular injection, in brain pond, locally, as scattered Agent, ointment or drops (including eye drops), including under cheek and sublingual, percutaneous, pass through nebulizer, or known in the art Other transfer modes.
" system gives ", " whole body gives ", " periphery gives " and " peripherally administered " means to give as the term is employed herein Selective ATP produces inhibitor/cyclodextrin complexes, so that it enters the system of patient, thus it is similar with other to stand metabolism Process.
As the term is employed herein " parenteral give " and " parenteral " mean and parenteral and topical is administered to Give pattern, generally by injection, and include, but not limited in intravenous, intramuscular, intra-arterial, sheath, in capsule, in socket of the eye, intraocular, the heart In, intracutaneous, intraperitoneal, through under tracheae, subcutaneous, epidermis, in joint, under capsule, cavum subarachnoidale, intraspinal tube and breastbone inner injection and Infusion.
In certain embodiments, pharmaceutical composition is through systemic delivery (for example, giving via oral or parenteral).At certain In other embodiments a little, pharmaceutical composition is by being directly injected into into tumour or being directly injected into blood supply into tumour (for example, artery or venous blood supply) localized delivery.In some embodiments, pharmaceutical composition is given by whole body and local Medicine transmits.For example, the experimenter suffering from tumour directly can be injected by combining with the pharmaceutical composition being orally administered to the present invention Composition containing compositions described herein enters tumour or the blood supply of tumour is treated.If used locally and systemically Give, administer locally to give with whole body before whole body gives simultaneously and/or occur after whole body gives.
In certain embodiments, the treatment method of the present invention, including treat carcinous or front-cancerous tumour, including give The compositions described herein that described experimenter combines with second medicine and/or therapy.So-called " with ... combination " refer to or Simultaneously, sequentially, or a combination thereof gives selective ATP and produces inhibitor/cyclodextrin complexes and one or more therapeutic agents person. Therefore, the experimenter that selective ATP produces the combination of inhibitor/cyclodextrin complexes and/or therapeutic agent is given, can be identical Time (i.e., simultaneously) or accept in different time (i.e., sequentially, with any order in identical sky or in different skies) Selective ATP produces inhibitor/cyclodextrin complexes, and one or more therapeutic agent as described herein, if two kinds of medicines Compound action realize in experimenter.When order of administration, described medicine each other can be the 1st, the 5th, the 10th, the 30th, the 60th, the 120th, the 180th, 240 minutes or longer time in give.In other embodiments, the medicine that gives of order can each other the 1st, the 5th, the 10th, the 15th, 20 or Give in more skies.
When combination gives, the valid density that various medicines cause particular biological to react can be less than each medicine individually Valid density when giving, if thus allow to reduce one or more medicine give as single medicine relative to medicine when institute Need the dosage of dosage.The effect of multiple medicines is it may be that but need not be adduction or synergy.Can repeatedly give medicine. In such combination treatment, when the therapeutic action of the medicine giving for the first time not yet disappears, sequentially, simultaneously or separately give with After medicine.
In certain embodiments, such method includes giving pharmaceutical composition, its comprise described herein with one Or multiple chemotherapeutics and/or anti-oxidant compounds, including chemotherapeutics described here, and other medicines known in the art The composition of combination.Conjoint therapy includes in such a way sequentially, simultaneously and separately, or jointly gives described composition, I.e. when giving compound subsequently, the therapeutic action of first the selective ATP inhibitor being given not yet is wholly absent.? In one embodiment, second medicine is chemotherapeutics.In another embodiment, second medicine is antioxidant chemical combination Thing.In another embodiment, second medicine is radiotherapy.In further embodiment, except described combination Beyond the region of objective existence, can also give radiotherapy.In certain embodiments, the pharmaceutical composition that second medicine can separate is joined jointly System.
In some embodiments, the pharmaceutical composition of the present invention will mix a certain amount of one or more things to be transmitted Matter, presents in an amount at least sufficient to pass to the therapeutic agent of the combination of bacterium or other materials as preventative or therapeutic treatment A part.The desired concn of reactive compound in the particle will depend on the absorption of medicine, inactivation and excretion rate and institute State the transfer rate of compound.It should be noted that dose value changes also with the seriousness of illness to be mitigated.Want further Understand that, for any specific experimenter, concrete dosage should give described group according to individual need with giving or monitoring The professional judgement of the personnel of compound, is As time goes on regulated.Generally, dosage will use well known by persons skilled in the art Technology measures.
Dosage can be every based on described composition or its reactive compound (selective depressant that for example, ATP produces) The amount of kg weight in patients.For example, the scope of the amount being encapsulated in composition therein or compound has been designed, including about the 0.001st, 0.01st, the 0.1st, the 0.5th, the 1st, the 10th, the 15th, the 20th, the 25th, the 50th, the 75th, the 100th, the 150th, the 200 or 250 every kg of the such composition of mg or more Weight in patients.Other amounts will be well known by persons skilled in the art and be readily determined.
In certain embodiments, the dosage (selectivity that for example, ATP produces of described composition or its reactive compound Inhibitor) will be generally in the about 0.001 every kg weight range of mg-about 250 mg, particularly at the about 50 every kg of mg-about 200 mg In weight range, and more particularly in the about 100 every kg weight ranges of mg-about 200 mg.In one embodiment, dosage It is in the about 150 every kg weight ranges of mg-about 250 mg.In another embodiment, dosage is the about 200 every kg bodies of mg Weight.
In some embodiments, described composition or its reactive compound (selective depressant that for example, ATP produces) Molar concentration in pharmaceutical composition will less equal than about 2.5 M, 2.4 M, 2.3 M, 2.2 M, 2.1 M, 2 M, 1.9 M、1.8 M、1.7 M、1.6 M、1.5 M、1.4 M、1.3 M、1.2 M、1.1 M、1 M、0.9 M、0.8 M、0.7 M、0.6 M, 0.5 M, 0.4 M, 0.3 M or 0.2 M.In some embodiments, described composition or its reactive compound (for example, ATP Produce selective depressant) concentration will less equal than about 0.10 mg/ml, 0.09 mg/ml, 0.08 mg/ml, 0.07 Mg/ml, 0.06 mg/ml, 0.05 mg/ml, 0.04 mg/ml, 0.03 mg/ml or 0.02 mg/ml.
Or, dosage can refer to described composition or its reactive compound (selective depressant that for example, ATP produces) Determination of plasma concentration.For example, maximal plasma concentration (C can be usedmax) and dense from the blood plasma of time 0-infinite (AUC (0-4)) Area under degree-time graph.The dosage of the present invention includes producing for CmaxValues above with AUC (0-4) and other dosage Those dosage, it causes the larger or smaller value for those parameters.
The actual dose level of the active component of the described composition of the present invention can change, and effectively realizes to specific to obtain The active component of the required therapeutic response of patient, composition, and mode of administration, and the amount nontoxic to patient.
The dosage level selecting will depend upon which that various factors includes particular therapeutic agent in preparation used, or its ester, salt or acyl The activity of amine, method of administration, administration time, the metabolism of discharge rate particular therapeutic agent or to be used, the duration for the treatment of, Other medicines, compound and/or the material being applied in combination with specific compound used, the age of patient to be treated, sex, body The factor known to medical domain such as weight, illness, general health and previous medical history.
There is the clinician of this area common skill or animal doctor is readily determined and issues required pharmaceutical composition The prescription of effective dose.For example, clinician or animal doctor can issue and/or give with the dosage lower than desired level for medicine The compounds of this invention of compositions, to realize required therapeutic action and to be gradually increased dosage, until it reaches required effect.
In general, the suitable daily dose of the compounds of this invention will be the change of the lowest dose level effectively producing result for the treatment of The amount of compound.Such effective dose will generally depend on above-mentioned factor.
If it is required, effective daily dose of reactive compound can be spaced at daylong appropriate time, optionally with single Dosage form, as the 2nd, the 3rd, the 4th, the 5th, 6 or more Asia-dosage separately giving give.
The administration correct time of any specific compound of most effective treatment will be produced and measure and will take in given patient Certainly in the activity of specific compound, pharmacokinetics and bioavilability, the physiological condition of patient (includes age, sex, disease Type and stage, general physical condition, the reactivity to the dosage of given medicine and type), method of administration etc..Presented herein Medication guide can be used to optimize treatment, for example, determines the Best Times and/or amount being administered, and it will need to exceed by monitoring Experimenter and the normal experiment of regulation dosage and/or time composition.
While experimenter accepts treatment, the health of patient can be by the scheduled time measurement one during 24-hour Individual or multiple index of correlation monitorings.All for the treatment of aspect, including be administered and the supplementing of preparation, amount, number of times, can be according to such The result of monitoring optimizes.Patient periodically can reappraise to determine improved degree, for the first time by measuring identical parameter Such reappraising usually occurs in the 4th weekend starting from treatment, and subsequently to reappraise during treating every 4-8 all Occur, then within every 3 months, carry out thereafter.Treatment can some months or even several years continuously, the minimum of one moon is to be typically used for The course for the treatment of of people.The amount of the medicine that regulation for example gives and administration number of times can reappraise based on these and carry out.
Treatment is available to be started less than the smaller dose of the dose,optimum of compound.Afterwards, the increment that dosage can be little increases Until reaching optimal result for the treatment of.
As described above, composition or its reactive compound (selective depressant that for example, ATP produces) can be with radiotherapy Combination gives.Radiotherapeutic dose,optimum can give experimenter as daily dose.Radiotherapeutic the suitableeest daily dose it may be that For example, from about 0.25-0.5 Gy, about 0.5-1.0 Gy, about 1.0-1.5 Gy, about 1.5-2.0 Gy, about 2.0-2.5 Gy, peace treaty 2.5-3.0 Gy.Exemplary daily dose it may be that for example, from about 2.0-3.0 Gy.For example, if the spoke to relatively low-dose for the tumour Penetrate resistance, then can give the radiation of higher dosage.The radiation of high dose can reach, for example, and 4 Gy.Additionally, at therapeutic process In the radiation accumulated dose that gives can be for example in the range of about 50-200 Gy.In an exemplary, at therapeutic process In the radiation accumulated dose that gives in the range of for example, from about 50-80 Gy.In certain embodiments, the dosage of radiation can be For example, the 1st, the 2nd, the 3rd, the time interval of 4 or 5 minutes when give, what wherein time quantum depended on radiation source gives rate.
In certain embodiments, the daily dose of the suitableeest radiation can give, for example, 4 or 5 days weekly, persistently about 4-8 week. In alternate embodiment, the daily dose of the suitableeest radiation can 1 week 7 days, every day gives, persistently about 4-8 week.Some embodiment party In case, the daily dose of radiation can give with single dose.Or, the daily dose of radiation can give as multiple dosage.Entering one In the embodiment of step, the dose,optimum of radiation can be than the higher radiation of the patient's tolerable dose based on basis every day Dosage.Therefore, the radiation of high dose can give patient, but gives with the less dosage of frequency.
Can be used for the radiation type for the treatment of of cancer to be well known in the art and include electron beam, from linear accelerator or put Penetrate high-energy photon, the proton of source such as cobalt or caesium, and neutron.Exemplary ionization radiation is the radiation of x-ray.
The method giving radiation is well known in the art.Illustrative methods includes, but not limited to outer light beam radiation, interior light Bundle radiation, and radiopharmaceutical.Outside in light beam radiation, linear accelerator is used to transmission height-energy x-ray to by cancer The body region of impact.Outside originating from health due to radiation source, outer light beam radiation can be used to uniform dose and control Treat large-area health.Internal radiation therapy, also referred to as brachytherapy, relate to the health that is radiated to transmitting high dose Privileged site.Two kinds of major type of internal radiation therapy include interstitial radiation, and wherein radiation source is placed on by shadow In the tissue ringing, and intracavitary irradiation therapy, wherein radiation source is placed on the very short inner body cavity of the distance with involved area In.Radioactive material also can be passed to tumour cell by being connected to tumor specific antibody.For internal radiation therapy Radioactive material be commonly included in Caplet agent, bead, line, in pipe, or implant.On the contrary, radiopharmaceutical is can per os Clothes, intravenous or be directly entered the unpacking radiation source that body cavity gives.
Radiotherapy may also comprise stereotactic surgery or stereotactic radiotherapy, and the wherein radiation of precise volume can use (it is that one was painted before radiation therapy for linear accelerator or gamma knife and 3 D stereo orientation conformal radio therapy (3DCRT) The area of computer aided therapy of the aspect graph of knub position processed) it is transferred to little tumor region.
The toxicity of this compound and treatment effect for example, can measure LD by cultivating or in animal used as test at cell50With ED50Standard pharmacological program determination.Preferably demonstrate the big composition treating index.In some embodiments, can measure LD50(lethal dose) the selective ATP inhibitor combination for cyclodextrin-encapsulating described herein can be relative to Without the selective ATP inhibitor of any cyclodextrin encapsulating, for example, at least 10%, the 20%th, the 30%th, the 40%th, the 50%th, the 60%th, the 70%th, the 80%th, 90%th, the 100%th, the 200%th, the 300%th, the 400%th, the 500%th, the 600%th, the 700%th, the 800%th, the 900%th, 1000% or more reduce.Similarly, may be used Measurement ED50(i.e., it is achieved the concentration of the half of symptom-maximum suppression) the selectivity for cyclodextrin-encapsulating described herein ATP inhibitor combination can be relative to the selective ATP inhibitor encapsulated without any cyclodextrin, for example, at least 10%, 20%、30%、40%、50%、60%、70%、80%、90%、100%、200%、300%、400%、500%、600%、700%、800%、900%、 1000% or more increase.Equally, similarly, IC can be measured50(i.e., it is achieved to the half-maximum cell toxicity of cancer cell or The concentration of Cell growth inhibition effect) and the selective ATP inhibitor combination for cyclodextrin-encapsulating described herein permissible It is relative to the selective ATP inhibitor encapsulated without any cyclodextrin, for example, at least 10%, the 20%th, the 30%th, the 40%th, the 50%th, the 60%th, 70%th, the 80%th, the 90%th, the 100%th, the 200%th, the 300%th, the 400%th, the 500%th, the 600%th, the 700%th, the 800%th, the 900%th, 1000% or more increase.Though So can use the compound demonstrating toxic side effects, it should be noted that design makes site needed for targeting compounds, to subtract The transmission system of few side effect.
In some embodiments, disclosed method produce in determination method growth of cancer cells at least about the 10%th, the 15%th, 20%th, the 25%th, the 30%th, the 35%th, the 40%th, the 45%th, the 50%th, the 55%th, the 60%th, the 65%th, the 70%th, the 75%th, the 80%th, the 85%th, the 90%th, 95%, or even 100% Suppression.
In any said method, give selective ATP and produce inhibitor/cyclodextrin complexes and give selective ATP Solid malignant before producing inhibitor/cyclodextrin complexes compares, and may result in the solid malignant of experimenter extremely Few about the 10%th, the 15%th, the 20%th, the 25%th, the 30%th, the 35%th, the 40%th, the 45%th, the 50%th, the 55%th, the 60%th, the 65%th, the 70%th, the 75%th, the 80%th, the 85%th, the 90%th, 95%, Or even 100% minimizing.
In some embodiments, the selective ATP of therapeutically effective amount produces inhibitor/being prevented property of cyclodextrin complexes Give, to prevent experimenter from forming solid malignant.
In some embodiments, experimenter is people.In other embodiments, experimenter is not people, and such as lactation is moved Thing.
The dosage range of people is can be used for being formulated for from the data that cell culture assay and zooscopy obtain.Any benefit Fill, or the dosage of any of which component alternatively, it is preferably located in and include ED50Circulation composition in the range of, with pole Even few does not has toxicity.Dosage can be dependent on formulation used and the method for administration of employing changes within the range.For this Bright medicine, therapeutically effective amount can start estimation from cell culture assay.Dosage can be prepared to reach to include in animal model Such as the IC being measured in cell cultivation50Circulating plasma concentration range.Such information can be used to measure more accurately Dosage useful in the mankind.Level in blood plasma can, for example, by high-efficient liquid phase color spectrometry.
G. kit
Selective ATP described herein produces inhibitor/cyclodextrin complexes and composition can be assembled into for treatment or pre- The kit of anti-disease, such as cancer or drug system.In some embodiments, 3-BrPA-cyclodextrin complexes and combination Thing can be used guard against or treat the solid malignant being caused by cancer.In general, the kit of the disclosure contains Or all of component, reagent, complementary goods etc. are with the method for implementation basis disclosure theme.Kit generally comprises answering of effective dose Compound is preventing, postpone, to reduce, or the disease (for example, solid malignant) that treatment is harmful.In one embodiment, medicine Agent box includes that at least one comprises selective ATP described herein and produces inhibitor/cyclodextrin complexes and/or combinations thereof Container (for example, carton box, glass bottle, bottle, pipe, or ampoule).Generally, compound and/or composition will be in one or more appearances Supplying in device, the compound that each container contains effective dose is so that solid malignant disappears, slows down, or is suppressed.
Therefore, in some embodiments, the theme of the disclosure provides a kind of kit, and it comprises to be encapsulated at least one At least one selective ATP in cyclodextrin carrier produces inhibitor.In other embodiments, kit also comprises to use bag At least one selective ATP being enclosed at least one cyclodextrin carrier produces a set of specification of inhibitor.
The selective ATP of separate storage produces inhibitor and cyclodextrin, then merges them before use and is probably needs. Therefore, in other embodiments also having, at least one selective ATP that kit comprises in a vessel produces suppression Agent and at least one cyclodextrin carrier in another container.
Example
Following example are included to be supplied to the representative embodiment party that those of ordinary skill in the art put into practice disclosure theme The guidance of case.According to the mean level of present disclosure and those skilled in the art, skilled artisans appreciate that, following example It is merely meant to exemplary, and it can be deployed in many changes, modification, and change is without departing from the model of presently disclosed theme Enclose.Following example provide by way of illustration and without limitation.
Embodiment 1: the material of embodiment 2-3 and method
A. the universal method of the beta-schardinger dextrin of synthetic modification
Succinyl group-beta-cyclodextrin is purchased from Sigma Chemical (St. Louis, MO, USA；Classification number 85990).Buy The beta-schardinger dextrin of unmodified and alpha-cyclodextrin (Sigma-Aldrich, St. Louis, MO).
But, it is possible to ambroin is acylated cyclodextrin.For example, the tosyl of 0.9 molar equivalent being used in pyridine 6 ' the primary hydroxyls to beta-schardinger dextrin (Sigma-Aldrich, St. Louis, MO) for the chlorine carry out mono-tosyl (mono- Tosylated), it to provide corresponding tosylate, by processing in acetone with sodium iodide, is translated into iodo-spread out Biological.It by iodo derivative with suitable amine through heating 8-12 h in 80 DEG C, is converted into 6 ' required aminocyclodextrin (Tang With Ng (2008)Nat. Protocol.3:691-697).By processing parent in DMF with the succinyl oxide of 0.9 equivalent Beta-schardinger dextrin, synthesizes 6 '-mono-succinyl group-beta-cyclodextrin (Cucinotta etc. (2005)J. Pharmaceut. Biomed. Anal.37: 1009-1014).Product precipitates in acetone and purifies through HPLC before use.
The pH scope with optimum stabilization is pH 4-9.
B. the general program of the compound of encapsulating is prepared
Prepare the 3-BrPA being encapsulated in succinyl group-beta-cyclodextrin in 1:1 ratio.By 3-BrPA (150 mg, 1 mmol) Join with aliquot (10 mg every time) in the agitating solution of succinyl group-beta-cyclodextrin (1,500 mg are in distilled water).Complete Become after adding, Solution Under Ultrasound Treatment 1 hour at room temperature.Then sonicated solution is allowed in 25 DEG C at constant temperature blending Shaking overnight in instrument (thermomixer), snap frozen being lyophilized in dry ice-propanone bath.
Similarly, prepare, in the ratio of 2:1, the 3-BrPA being encapsulated in succinyl group-beta-cyclodextrin.By 3-BrPA (166 Mg, 1 mmol) join succinyl group-beta-cyclodextrin (918 mg are in 20 ml distilled water) with aliquot (10 mg every time) In agitating solution.After the addition was complete, Solution Under Ultrasound Treatment 1 hour at room temperature.Then sonicated solution is allowed to In 25 DEG C of shaking overnight in constant temperature blending instrument (thermomixer), snap frozen being lyophilized in dry ice-propanone bath.
Additionally, prepare, in the ratio of 1:1, the 3-BrPA being encapsulated in alpha-cyclodextrin (see following structure).By 3-BrPA (166 mg, 1 mmol) joins alpha-cyclodextrin with aliquot (10 mg every time), and (972 mg, 1 mmol are at 10 ml distilled water In) agitating solution in.After the addition was complete, Solution Under Ultrasound Treatment 1 hour at room temperature.Then sonicated solution quilt Allow in 25 DEG C of shaking overnight in constant temperature blending instrument (thermomixer), snap frozen being lyophilized in dry ice-propanone bath. Use non-GRAS and GRAS version by similar result.
Alpha-cyclodextrin structure
Determine surprisingly, substitute its α-D-pyrans one or more by the ionogen producing negative electrical charge (anion) The modification cyclodextrin of the one or more hydroxyls on glucoside unit, has the ionizable of generation positive charge (cation) than those The cyclodextrin of group or the cyclodextrin of unmodified, the α-of such as unmodified or beta-schardinger dextrin preferably stablize 3-halogen acetone Acid.Also determine surprisingly beta-schardinger dextrin encapsulating in protection with the 3-BrPA of form stablizing 3-BrPA, particularly in vivo Curative effect and also curative effect aspect is significantly better than alpha-cyclodextrin in vitro.
Additionally, prepared Cell culture invitro and the treatment of internal mouse and as above with as follows to succinyl group-beta-cyclodextrin Carry out described in encapsulating 3-BrPA, use the beta-schardinger dextrin being commonly referred to be safe (GRAS) version (for example, to have such as followingization Hydroxypropyl-the beta-schardinger dextrin of the 3-5 substitution level shown in form) 3-BrPA that encapsulates, and result is similar to succinyl Those results that group-beta-cyclodextrin encapsulating 3-BrPA describes.
GRAS hydroxypropyl-beta-schardinger dextrin structure
C. Cell culture invitro
3-BrPA and beta-schardinger dextrin (medium) are purchased from Sigma Chemical (St. Louis, MO, USA).For viablity Measure, by MiaPaCa-2 and Suit-2 cell with 5 x 103The density of cell per well, by being inoculated in 96-orifice plate in triplicate In.After 12 hours, with 3-BrPA, the CD-3BrPA (0-150 μm) increasing concentration and mordanting cell.Intracellular ATP water The flat scheme according to manufacturer, uses cell titer-Glo luminescent cell viablity (Cell Titer-Glo Luminescence Cell Viability) measure kit (Promega, Durham, NC, USA) mensuration.Measurement after treatment 24 hours and Within 72 hours, carry out.
D. mouse interior therapeutic
15 animals accept mg/kg β-CD-3BrPA every day 5 (with the ratio of 1:1) (N=10) or vehicle control at random altogether (N=5) injection.Baseline biodiversity resources confirms tumor growth (5 generations showing in figures 4 and 5 in all animals Table animal).After intraperitoneal injection 2 weeks, all animals stand follow-up imaging.Show bioluminescence with the animal of mordanting Strongly increasing of signal, represents tumour progression.
The mechanism criterion of the scheme according to approval use Male athymic nude mice (20-25 g, 4 week old, Crl:Nu-Nu, Charles River Laboratories, Wilmington, MA, USA).Mouse is maintained at the layer under constant temperature and constant humidity In flow chamber, freely give food and water.Under the control of EF-1 α promoter, use with luciferase-aminoglycoside phosphorus The MiaPaCa-2 clone of acid transferase fusion stable transfection.Mouse was sucked by isoflurane before operation and treatment Anaesthesia is anaesthetized.Manufacture a little left veutro otch and pancreas is taken out in abdomen.By injection 1-2 x 106 MiaPaCa- 2 cells enter pancreas afterbody, generate Vipoma in situ.It is injected through fluid bubble in pancreas under the successful capsule of tumour cell Outward appearance is identified without intraperitoneal leakage.
For biodiversity resources (BLI), carry the D-luciferin with 150 mg/kg for the anesthetized mice of TIS (Gold Biotechnilogy, St Louis, MO, USA) intraperitoneal injection simultaneously uses IVIS 100 after 5 minutes (Xenogen Corp, Alameda, CA, USA) optical imagery.Represent the pseudocolour picture of the spatial distribution of the photon detecting As being superimposed on gray scale photographs.Use image software alive (Living Image software) (Xenogen Corp.) after exposing 10 seconds, quantitative (p/s/cm2/Sr) to signal strength signal intensity with ROIs.After tumour is implanted the 7th, the 14th, the 21st, 28 and 35 It carries out imaging.
After tumour is implanted after 1 week, after using BLI to confirm tumor growth in each animal, all of animal is divided at random Be 3 groups with via intraperitoneal injection every day (volume injected, 500 l/mouse/sky；Dosage, 5 mg/kg) accept or 3- BrPA, CD-3BrPA or medium.Injection solution, by being dissolved in described chemicals in phosphate buffered saline (PBS), is regulated to pH 7.4 prepare.Animal 1 time and every 4-6 hour observation 1 time after each subsequent injections is observed per hour during starting injection. Record any change in the overall clinical condition of all treatment groups.
After last BLI imaging in 24 hours, dislocation of cervical vertebra is used to put to death animal.Open whole belly and use spleen and The monoblock of pancreas is extracted and is obtained tumour.Tumor sample uses 4% paraformaldehyde to fix 72 hours, with FFPE and cut into slices.Tissue Learn section dye through h and E (H&E) and seeked advice from explanation by virologist.
Embodiment 2: the external impact on human pancreatic cancer cell for the 3-BrPA of cyclodextrin encapsulating
Two clones, i.e. MiapaCa-2 and Suit-2 to human pancreas cancer, test them to 3-BrPA and CD-3-BrPA Reaction.MiaPaCa-2 is derived from the human adenocarcinoma of local infiltration and forms typical entity tubercle in pancreas.It is known to several Individual standard care anticancer, including gemcitabine demonstrates significant resistance.Suit-2 is derived from and divides from metastatic hepatic neoplasms From the pancreatic neoplasm of Highly invasive.It has the phenotypic characteristic of Highly invasive, such as aggressive and animal migration.Cell is anti- Should or Cells viability use standard ATP vitality test method measure.Each experiment repeats at least twice, uses three parts to repeat biology Sample.
(traditional 3-BrPA in phosphate-buffered saline does not has any cyclodextrin bag to Fig. 2 A-2B display 3-BrPA Envelope or compound are formed) or the β-CD-3-BrPA (3-of the succinyl group-beta-cyclodextrin encapsulating in phosphate-buffered saline BrPA) impact after processing 24 hours on MiaPaCa-2 cell (Fig. 2 A) and Suit-2 cell (Fig. 2 B).Data display β- The dose dependent that the Cells viability that CD-3-BrPA is processed compares with the cell that 3-BrPA is processed reduces.In addition, multiple drug-resistance MiaPaCa2 cell is found more sensitive to 3-BrPA/CD-3-BrPA comparison Suit2 cell.(beta-schardinger dextrin is at phosphoric acid for medium In salt-BS, without any 3-BrPA) itself do not cause any toxicity.
Fig. 3 show 3-BrPA (traditional 3-BrPA in phosphate-buffered saline, do not have any cyclodextrin encapsulate or Compound formed) or β-CD-3-BrPA (in phosphate-buffered saline succinyl group-beta-cyclodextrin encapsulating BrPA) exist Impact on MiaPaCa2 cell after processing 72 hours.MiaPaCa-2 cell shows, even if with the CD-3-of ~ 50 uM concentration BrPA is processed 72 hours, and viablity is also significantly lost, and processes 24 hours (Fig. 2 A) with identical concentration, does not significantly survive Power is lost.Therefore, the longer duration (48 hours) that employing is processed, the CD-3-BrPA of at a fairly low concentration (50 uM) be enough to open Kinetocyte dead (~ 50% is dead) (Fig. 3).
Embodiment 3: internal effect in the mouse model of human pancreas cancer for the 3-BrPA of material and cyclodextrin encapsulating
The athymic mouse model of human pancreas cancer is used in studying in vivo.Human pancreatic cancer cell, MiaPaca-2, stablizes table Reach luciferase gene, implanted in pancreas in situ.Tumor growth and reaction are monitored by biodiversity resources.
Table 1 is described in the clinical sign observed in the tumor animal of 3-BrPA or the CD-3-BrPA treatment of height-dosage Or symptom.These symptoms with without bias and blind-research (unbiased and blind-study) mode record.These diseases Shape is at dosage > 5 mg/kg body weight and concentration ~ 3.5 mM (higher than recommend therapeutic dose) when be observed.Unwanted face Bed S or S is found in the mouse treated by 3-BrPA, and these S or Ss considerably less than use CD-3-BrPA The mouse for the treatment of, represents that cyclodextrin carrier protection experimenter avoids the side effect of 3-BrPA molecule.
Table 1
With succinyl group-beta-cyclodextrin-3-BrPA treatment animal show to biodiversity resources completely or nearly-completely Tumor response (Figure 4 and 5).Subsequently, put to death animal and confirm bioluminescence result (Fig. 4) with autopsy.Result instruction CD-3BrPA is very Still retain its active anticancer to after forming compound with CD.
Carry out the histopathological analysis of MiaPaCa-2 tumour in situ.Fig. 6 shows, h and E (H&E) dyes Tumour is shown in control group and is not changed in, and shows extensive central necrosis from the tumour of the animal results for the treatment of and dissociate The region of tumor tissues.
Accordingly, it is determined that cyclodextrin complexes forms the anticancer property not affecting 3-BrPA, as from vitro and in vivo number According to confirmed.Equally, the activity of 3-BrPA can be kept or protected by CD, until it is passed or is distributed to target organ or swollen Knurl.Additionally, CD-3-BrPA gives animal causes the toxicity more less than alone 3-BrPA or related-clinical sign.
Embodiment 4: the material of embodiment 5-6 and method
Use following material and experiment described in embodiment 2-3 for the method extension, to advance the result obtaining from which.Example If ductal pancreatic adenocarcinoma (PDAC) ranking is the 4th modal reason (Siegel etc. (2014) of world's cancer related mortalityCA Canc. J. Clin.64:9-29).Owing to the Most patients stage late is just diagnosed, treatment option remains limited , and prognosis is depressing, 5-viablity is less than 5% (Hidalgo (2010)New Engl. J. Med. 362: 1605-1617).In past 20 years understand cancer of pancreas tumour occur and disease development during bring great enter Exhibition, it can be considered a different and multifactorial neoplastic process (Hidalgo (2010) nowNew Engl. J. Med. 362:1605-1617；Hanahan and Weinberg (2011)Cell144: 646-674).Pancreatic tumor tissue is By several uniquenesses, cell and non-cell constituents include rich in collagen, vascularization difference, highly anoxic, Non-cancerous matrix composition (Chu etc. (2007)J. Cell. Biochem.101:887-907；Mahadevan and Von Hoff (207)Mol. Canc. Therapeut.6:1186-1197).The anticancer that these features are applied with the most frequently used system, such as gemcitabine Related (the Muerkoster etc. (2004) of prominent chemoresistanceCancer Res.64:1331-1337；Yokoi and Fidler (2004) Clin. Canc. Res.10:2299-2306).It should be noted that the energetic supersession of change is nearest It in the organisation of the organisation being added to tumor microenvironment and is regarded as now " mark " of cancer of pancreas (Hanahan and Weinberg (2011)Cell144:646-674；Guillaumond etc. (2014)Arch. Biochem. Biophys545:69-73.Oxygen independently depend on as cancer cell energy supply main shaft glycolysis for a long time always by It is referred to as " fertile berg's effect (Warburg effect) "；But, this situation is not yet clinically used for therapeutic purposes (Warburg Deng (1927)J. Gen. Physiol.8:519-530；Ganapathy-Kanniappan and Geschwind (2013)Mol. Cancer12:152).3-bromo acetone acid (3-BrPA), a kind of highly effective enzyme glyceraldehyde phosphate-3-dehydrogenase (GAPDH) micromolecular inhibitor, is unique obtainable anti-glycolysis drug candidate, and it can be optionally through a carboxylic Acid transporter albumen 1 (MCT1) enters cancer cell (Ganapathy-Kanniappan etc. (2009)AntiCancer Res. 29: 4909-4918；Birsoy etc. (2013)Nature Genet.45:104-108).The Anti-tumor effect of 3-BrPA is by extensively Study generally and confirm in several murine tumors models, in the environment of local-regional therapy, or passing through tumour-confession Blood artery or direct intra-tumoral injection transmit (Ota etc. (2013)Target. Oncol.8:145-151；Geschwind etc. (2002) Canc. Res.62:3909-3913).But, due to the alkylation characteristic of 3-BrPA, when with therapeutic dose system During transmission, it has shown that significant toxicity, and this may hinder clinical development in cancer patient for this medicine in turn With use (Chang etc. (2007)Acad. Radiol. 14:85-92；Cao etc. (2008)Clin. Canc. Res. 14: 1831-1839)。
A. antibody, reagent and kit
Use following first antibody: rabbit anti-MM P-9 polyclonal antibody (pAB) #3852 (Cell Signaling), DAPI # D1306 (Invitrogen), Alexa Fluor 568 phalloidine (Phalloidin) #12380 (Life Technologies), GAPDH (14C10) monoclonal AB (mAB) Alexa Fluor 488 conjugate #3906 (Cell Signaling), GAPDH pAB #sc-47724 (Santa Cruz), Caspase-3 pAB #9661 of cracking (Cell Signaling), MCT-1 pAB #sc50324 (Santa Cruz), and Ki-67 kit/antibody (Dako Inc.).Use following SA: #7074 (Santa Cruz), the anti-rabbit igg (H+ that goat anti-rabbit igg HRP-puts together L)、F(ab')2Fragment PE conjugate #8885 (Cell Signaling), and goat anti-mouse IgG-FITC #sc2010.Make Use following chemicals: 3-bromo acetone acid (3-BrPA, Sigma Aldrich), GEMCITABINE HYDROCHLORIDE (LC Laboratories), succinyl group-beta-cyclodextrin (β-CD, Sigma Aldrich), and D-luciferin sylvite (Gold Biotechnology, St Louis, MO, USA).Use following cell culture reagent: RPMI-1640 (Life Technologies), MEM (Life Technologies), hyclone (FBS, Thermo Scientific), mould Element/streptomysin (Sigma Aldrich), the big rat-tail of collagen I (BD Biosciences, #354326), and controlled atmosphere chamber (Plas. Labs).Use following aggressive measure reagent: matrigel basement membrane matrix (BD Biosciences) and matrigel Invasion and attack cell transwell polycarbonate membrane inserts (Corning).Use following kit: CellTiter-Glo luminous thin Born of the same parents' viablity measures kit (Promega), luciferase reporter gene measures kit (Promega), 2D quantitative reagent Box (GE Healthcare), histostain add the third generation (3rd gen) ICH detection kit (Invitrogen), and Diff quik staining kit (Polysciences Inc.).Use imaging below equipment: Zeiss 700 LSM copolymerization burnt aobvious Micro mirror, Olympus IX81 inverted microscope, Eclipse TS100 inverted microscope (Nikon), and IVIS200 (Xenogen Corp., Alameda, CA)。
B. complex formulation and nuclear magnetic resonance (NMR) spectroscopic methodology
For preparing the 3-BrPA being encapsulated in β-CD, 3-BrPA (166 mg, 1 mM) is joined with aliquot (10 mg every time) In the agitating solution of β-CD (918 mg are in 20 ml DI water).After the addition was complete, in 50 DEG C of Solution Under Ultrasound Treatments 1 hour. Then sonicated solution is allowed in 25 DEG C of shaking overnight in constant temperature blending instrument (thermomixer), at dry ice-the third Snap frozen being lyophilized in ketone bath.Lyophilized compound is used to study for further biology and biophysics as former state.1H NMR experiment is carried out on Bruker Avance spectrometer with 400 MHz.H NMR spectroscopy is with 99.9% D2O record and with relative to Every million low numbers report of tetramethylsilane (TMS).β-CD is independent, 3-BrPA independent, or 3-BrPA and β-CD compound 10 mM solution, with 1% DSS (3-(trimethyl silyl)-1-propane sulfonic acid, sodium salt；Sigma Aldrich) as interior Prepared by ministerial standard.In 25 DEG C, by 32 frame scan record spectrums.The high field displacement of methene proton (0.1 ppm) is seen after compound Observe (see Fig. 7).
C. monolayer cell culture and viablity measure
Two kinds of human pancreas's gland cell systems,lucMiaPaCa-2 is (with luciferase-aminoglycoside phosphotransferase fusion Stable transfection, is provided by Dr. Phuoc T. Tran is friendly) and Suit-2 (by Dr. Shinichi Ota, Japan give) Cultivating in RPMI or MEM culture medium respectively, both culture media supplemented have 10% FBS and 1% Pen .-Strep.By making With luminescence-base kit (CellTiter-Glo, Promega) and multi-tag 96-orifice plate (Costar), three phosphorus in quantization cell Adenosine monophosphate (ATP) level, measures the effect to Cells viability for the different medicines.This fluorescence-base kit is used to exist The accuracy of the viablity measurement in lucMiaPaCa-2 cell measures examination with can be used repeatedly luciferase reporter gene Agent box (Promega) confirms.In short, by 5 x 103Cell is by triplicate inoculation and at normal oxygen or anoxic (1% O2-level, Use CO2With nitrogen in controlled atmosphere chamber inner equilibrium) under the conditions of cultivate 72 hours.Make free 3-BrPA, 1:1-β-CD-3-of specified amount BrPA or the β-CD as comparison is dissolved in PBS and joins process 24 hours in culture medium.For the experiment using gemcitabine, Cell was cultivated 24 hours before being exposed to medicine 72 hours.Cells viability measures according to the scheme of manufacturer.
D. 3D organotypic cell cultivation, imaging and immunofluorescence
Use the 3D organotypic cell based on collagen 1 to cultivate, with analog cell outer-environment of matrix (ECM)-enrichment test 3- Effect (the Cheung etc. (2013) to tumor invasiveness for the BrPACell155:1639-1651；Nguyen-Ngoc and Ewald (2013) J. Microscop.251:212-223).Especially, a kind of initially by 25 l 10x DMEM and 217 l collagens The collagen solution that I (3.83 mg/ml) forms is prepared on ice.Reach pH by dropping NaOH (Sigma Aldrich) =7.0 regulate pH value.Then use DMEM F12/GlutaMAX (Life Technologies) that collagen I is diluted to 3 The ultimate density of mg/ml.In the bottom in each hole of uncovered glass bottom 24-orifice plate (InVitroScientific), use 15 L collagen solution creates bottom, then makes it be polymerized at least 1 hr in 37 DEG C.Remaining collagen solution is maintained at 3-5 hour on ice, To allow initial polymerization.Make 65 x 10 altogether3 lucMiaPaCa-2 or 45 x 103Suit-2 cell is resuspended in 150 l In the collagen solution of volume.By creating the decline of 0.5 cm height, cell suspending liquid is placed on the top of preheating low layer.Make glue Former-cell suspending liquid is polymerized 1h in 37 DEG C, covers (Nguyen-Ngoc and Ewald (2013) with cell culture medium subsequentlyJ. Microscop. 251:212-223)。
3D organelle or once or process in order.For single treatment, make the cell of embedding before treatment, often Oxygen or anoxic (1% O2-level is indoor at controlled atmosphere) under the conditions of cultivate 5 days.At the 5th day, culture medium with containing 1:1-β- The culture medium of CD-3-BrPA/3-BrPA/ β-CD is replaced, and cultivates cell 24 hours together with the medicine of each concentration.For making With the experiment of gemcitabine, cell is made to grow 48 hours and cultivate other 72 hours together with medicine before treatment.Use Ji Xi The initial experiment of his shore does not show any effect after 24 hours, thus determines that the incubation time 72 following most common report is little When.Carry out every other day with respective dosage by the continuous processing of 3-BrPA, continue 1 week and with bright-field microscope (Olympus), with 40x enlargement ratio, with 1.3 NA oil eyepiece evaluations.Use Hamamatsu photoelectricity C9100-02 EMCCD camera (Hamamatsu Photonics C9100-02 EMCCD camera), obtain image by SlideBook 5.0 program.
The quantization that the upper finding Cells viability with biodiversity resources (BLI) such as in vitro observed by microscope compares. For latter measurement, cover the 500 ul luciferase substrate that the cell culture medium of 3D organotypic cell cultures is used in PBS (D-luciferin, sylvite, Life Technologies, 20 mg/ml) replace.After exposing 5 minutes, plate is positioned and collection figure As (Xenogen Ivis Imaging System 100).Launched by photon and determine signal strength signal intensity (counting) and closing entirely Measurement (Living Image Software, PerkinElmer) in the area-of-interest (ROI) of 3D organelle.
Use immunofluorescence micro-checking mirror microcosmic and BLI result.3D organelle use 4% formaldehyde fix and in-80 DEG C with OCT compound (Tissue Tek) cryofixation (cryofixed).Sample is cut into 100 m slabs in-20 DEG C.OCT PBS is used to wash twice, 10 mins every time.Before dyeing, section permeates 30 mins with 0.5 % Trizol 100 in PBS And use PBS to wash twice, 10 mins every time.After closing 2 hours with 10% FBS in PBS, make sample and first antibody (Alexa Fluor 568 phalloidine, Invitrogen, 1:100；GAPDH Alexa Fluor 488 conjugate, Cell Signalling, 1:800；Caspase-3 of cracking, Cell Signalling, 1:500；HIF-1 α 1:50) one Arise from room temperature (RT), light protection lower cultivation 1 hr.The first antibody puted together for non-, is also adopted by other and phycoerythrin (PE) SA one of-or fluorescein isothiocyanate (FITC)-put together arises from the RT cultivation of lower 1 hour.PBS is used after this Wash twice, every time 10 mins.DAPI the concentration of 300 ng/ml be used as counterstain (counter stain) and with put together Antibody be added simultaneously in sample.Sample is sealed by antifading mounting medium (antifading mountant) and uses lid glass Piece covers.Confocal fluorescent microscope is with 40x enlargement ratio, with 1.4 NA oil eyepieces and 63x enlargement ratio, with 1.4 NA oil mesh Mirror is carried out.Image Zen2012 software (Carl Zeiss) is analyzed.Excite and launch wavelength and recommended by conjugate manufacturer Those wavelength.For example, 555 nm are used to excite phalloidine (phalloidin) and PE-conjugate, 488 nm quilts It is used for Alexa Fluor 488, and FITC-conjugate excites, and 405 nm are used to excite DAPI.? Between 555 and 1000 nm to red fluorescence inspection launch, and 490 nm and above to green fluorescence inspection launch.When using respectively When redness or green fluorescence imaging, DAPI is transmitted in 490 below nm or 529 below nm capture.
E. matrigel invasion mensuration, zymography and Western blot
3-BrPA suppression tumor invasion ability use matrigel invasion assay, and gelatin zymography research (Hu and Beeton (2010) J. Visual. Exp.45:2445；Scott etc. (2011)J. Visual. Exp. 58: e3525).In order to matrigel invasion measures, prepare a kind of buffer solution that is coated containing 0.01 M Tris and 0.7% sodium chloride and be used in combination Dilute matrix gum base counterdie (BD Biosciences, #356234) to 300 ug/ml.Subsequently, Boyden room (Transwell, Corning；6.5 mm-diameters, 8 um apertures) it is merged in 37 DEG C of storages 2 with 100 l matrigel solution bags Hour is to allow gelatine.About 7.5 x 104Cell is resuspended in the culture medium without FBS of 500 l and insert is arrived in tiling In upper chamber, then put it in the 24-orifice plate of a culture medium containing FBS containing 750 l.Cultivated in 37 DEG C After night, join the different amounts of 3-BrPA being dissolved in PBS in upper chamber.After 48 hours, remove from matrigel with cotton swab Non-aggressive cell.It is fixedly attached at the cell of the invasion and attack of infiltration insert bottom side and with Diff Quik staining kit (Polysciences Inc.) dyes.Use colored inverted microscope (Eclipse TS 100), amplify with 4x, 10x and 20x Multiplying power carries out light microscope methods.By measuring the area of staining cell after treatment with 10x enlargement ratio, and untreated Sample compares, and the invasion and attack to cell quantify.
Carry out zymography to measure to measure the activity of the MMP-9 of secretion.Therefore, 4 x 106 Suit-2 cell and 2.5 x 106 lucMiaPaCa-2 cell is seeded in 75 cm2In-flask and in 37 DEG C, overnight incubation under conditions of normal oxygen.Next day, Add the fresh culture medium without FBS of the 3-BrPA containing variable concentrations and cell is cultivated other 24 hours.Subsequently, in collection Clear liquid, filtration, and use 2D Quant kit (GE Healthcare) to measure final protein concentration.After regulation concentration, Each sample is loaded in two 10% gelatin zymography gels (Novex, Invitrogen).After electrophoresis, coagulate at two Protein in one of jelly is by renaturation and allows in 37 DEG C that enzymatic digestion is overnight in development buffer.Gel is with 4 part 0.1% Coomassie brilliant blue (Coomassie Brilliant Blue) dyes 24 hours in 1 part of 100% methyl alcohol and is washed by dilution (DI) Wash until the region digesting is detected as white band.Western blot analysis uses duplicate gel to carry out.Protein quilt Trace is on PVDF-film and use 5% skimmed milk to close in 1x TBS and 0.1% tween/DI (TBST).First anti-MM P resists Body (Cell Signaling) uses and in 4 DEG C of overnight incubation with 1:1000 thinner ratio, then cultivates HRP-at room temperature and puts together SA (Santa Cruz) 1 hr.HRP provides a kind of electrochemical luminescence signals (ECL Kit, GE Healthcare), it uses ImageJ 1.46r software (Wayne Rasband, National Institute of Health) Analyze and be used for quantifying signal strength signal intensity by alternative line integration.
F. original position animal xenograft thing
According to approved animal protection and mechanism guidance (the institutional guidelines under using the committee Approved Animal Care and Use Committee) scheme, use Male athymic nude mice (body weight, 20-25 g； 4 week old；Crl:NU (NCr)-Foxn1nu；Charles River Laboratory, Germantown, MD, USA).Mouse It is maintained in the Laminar Flow Room of stationary temperature and humidity, freely give food and water.It is suspended in 50 l PBS by implanting 1.5 x 106 lucMiaPaCa-2, to the afterbody of anesthetized mice pancreas, generates xenograft tumor in situ.For accomplishing this Point, mouse be placed in a clean anesthesia induction room (oxygen flow velocity, 1 liter/min；Isoflurane concentration 3-4%).Righting After reflection loses, animal is placed on operation table top, wherein nose cone be used to maintain anesthesia (oxygen stream, 0.8 liter/min；Different Fluothane concentration, 1.5-2%).Manufacture one little, left veutro otch, and pancreas by external to use 30G Hamilton syringe (Hamilton syringe) injects cell solution.Identified under successful capsule by the outward appearance of the liquid bubble not having intraperitoneal to leak Injection in pancreas.(Kim etc. (2009) sewed up by double-deck non-absorbable suture material in abdominal cavityNat. Protocol. 4: 1670-1680)。
G. biodiversity resources and ultrasonic imaging
Tumour viablity confirms for the 7th day after operation is implanted via vivo biodistribution luminescence imaging (BLI).The tumor-bearing mice warp of anesthesia Intraperitoneal injection D-luciferin 150 mg/kg after 5 minutes, uses IVIS 200 system (Xenogen) optical imagery.Represent The pseudo color image of the spatial distribution of photon is superimposed on the gray scale photographs of previously acquisition.Create area-of-interest (ROI) to include optical tumor image.After 10-second exposure, use image alive (Living Image) software (Xenogen), Signal strength signal intensity is quantified in ROI with photons/second/centimeter/surface of sphere (p/s/cm2/Sr).Additionally, using After little-animal ultrasound imaging (USI) is processed, it was demonstrated that the growth in situ of tumour.In short, anesthetized mice stand by application have The MS-550D MicroScan transducer probe (having the broadband of 22-55 MHz) of 40 MHz, uses VEVO2100 (Visual Sonics Inc., Toronto, Canada, provided by Dr. Harry C. Dietz is friendly) inspection.Tumor-localizing makes Confirm (Ota etc. (2013) with the pinnacle of left kidney and the tail point of spleen as anatomic landmarkTarget. Oncol.8:145-151； Tuli etc. (2012)Translat. Oncol. 5:77-84)。
H. therapeutic scheme and image are followed up a case by regular visits to
Suffer from the animal of tumour, as confirmed by both LI and USI, be randomized into 4 groups: 1 (N=21 animal) connects group By intraperitoneal injection β-CD-3-BrPA compound every day, (with 1:1 mol ratio, 5 mg/kg 3-BrPA are at 26 mg/kg β-CD In, it is dissolved in 500 l salt solution), group 2 (N=7 animals) accept intraperitoneal injection gemcitabine, and (150 mg/kg are dissolved in 200 L salt solution, 3 injection/weeks, as generally reported in the literature, such as Liau and Whang (2008)Clin. Canc. Res.14:1470-1477；Larbouret etc. (2010)Annal. Oncol.21:98-103), organize 3 (N=7) to accept Intraperitoneal injection β-CD every day (26 mg/kg β-CD, be dissolved in 500 l salt solution), and group 4 (N=7 animals) accept every day The single 3-BrPA of intraperitoneal injection (5 mg/kg are dissolved in 500 l salt solution).All animals are controlled through the free of discontinuities of surrounding by a definite date Treat.The 21st, the 14th, the 7th, after injecting for the first time the gather BLI in 28 days.The 28th day or execution when dying at last imaging after date Animal.
I. immunohistochemistry
Putting to death after animal, obtaining tumour, fixing the period of at least 72 hours by 4% formalin, embedding for exempting from paraffin Epidemic disease tissue chemical analysis.The haematine of slide and eosin (H&E) dyeing establishing criteria scheme, for example at Casadonte and Caprioli (2011) Nat. Protocol.Those described in 6:1695-1709 are carried out.The thick tumor biopsy of 18 m It is colored, for following purpose: use Histostain to add third generation Gen IHC detection kit (Invitrogen), with And Ki-67 kit (Dako Inc.) detection GAPDH, MCT-1, Caspase-3 of cracking, and Ki-67.Especially, sample Product use dimethylbenzene to dewax and use the ethanol dilution factor series of reduction rehydrated.After being washed with deionized, in 95 °, make sample Product permeate 40 minutes in the recovery solution of the boiling containing citric acid (Dako).Make sample be cooled to RT and with 2 (altogether ~ 100 ul) peroxidase quencher solution cultivates 5 min together, closes 20 mins.With first antibody (GAPDH, 1:500； MCT-1, 1:250；Ki-67 and HIF-1 α；1:50, the Caspase-3,1:1,500 of cracking；In PBS) cultivation exist Under RT, at moist indoor generation 60 mins.By biotinylated secondary antibody and streptavidin-peroxidase Conjugate joins in sample with the order of 10 min every time.Make 26.5 ul 3,3 '-benzidine (DAB) chromophories and 1 Ml DAB substrate buffer solution is sufficiently mixed and through 5 mins, 100 ul is added each sample.Haematine is used as counterstain.Cultivate Step is followed by wash with distilled water and wash twice with PBS, 2 mins every time.The antifading mounting medium of sample (antifading mountant) seals and covers with cover glass.To the scanning of all of slide and with 20x enlargement ratio, use High resolution A perio®Scanning system (Vista, California, USA) digitizes.Then Aperio image instrument is used®Soft Part (Aperio ImageScope®Software) digitized slide is assessed.The histotomy that Ki-67-is dyeed, with 10x observes 5-10 the visual field altogether, the number of record Ki-67-positive cell, and the sum of cell is to calculate Ki-67 mark Index (formula: index [%]=[positive cell number/total cell number r] x 100).
J. statistical data analysis
All of experiment independently carries out and repeats at least 3 times.The data standard error of mean value ± average from experiment Difference is summed up.The statistics of data set compare with Student ' s t-inspection and unidirectional ANOVA inspection carry out.Animal is reported BLI signal strength signal intensity be normalized and be reported as the multiple based on baseline value.
Embodiment 5: β-CD-3-BrPA is shown in the strong anti-cancer effect during 2D and 3D cell is cultivated and targeting metabolism subtracts The aggressive potentiality of few cancer cell
After compound (Fig. 7) between NMR-spectra 3-BrPA and β-CD, the microcapsule formulation of medicine is used for further Experiment.3-BrPA (β-CD-3-BrPA) for evaluating microencapsulation realizes that dose-dependency ATP exhausts the effect with cell death Power, uses two kinds of human pancreatic cancer cell.MiaPaCa-2 is derived from PaCa (PDAC) and Suit-2 is derived from different trouble Metastatic PaCa (the Kitamura etc. (2000) of personClin. Exp. Metast. 18:561-571)。β- The dose-dependency effect of CD-3-BrPA and free 3-BrPA, and gemcitabine compares, and β-CD is with comparing.Due to Anoxic is usually relevant with the chemoresistance of PDAC, and anoxic exposes and is added in experimental design (Yokoi and Fidler (2004)Clin. Canc. Res.10:2299-2306；Kasuya etc. (2011)Oncol. Rep.26:1399-1406；Onozuka Deng (2011)Canc. Sci.102:975-982；Zhao etc. (2014)Canc. Res.74:2455-2464)).Have been found that β-CD-3-BrPA and free 3-BrPA is at normal oxygen (50-75 M), and demonstrates similar under the conditions of anoxic (12.5-25 M) Cytotoxicity feature, and it is interesting that more sensitive (Fig. 8) to medicine when anoxic.The clone individually processing with β-CD exists Whole experiment is survived, completely even when being exposed to high concentration.To Suit-2 cell observation to similar result, But the difference between normal oxygen and anoxia condition is less obvious (Fig. 8).When evaluate gemcitabine effect when, MiaPaCa-2 and The IC of Suit-2 cell50It is little to reach under the condition (0.1 M) of normal oxygen, do not have concentration to reach to kill completely, and anoxic Seem to add the resistance to medicine.
For checking effect in the environment rich in ECM for the β-CD-3-BrPA, willlucMiaPaCa-2 cell is at 3D collagen 1 Matrix is cultivated and processes with the β-CD-3-BrPA of single dose, free 3-BrPA or β-CD (as comparison).BLI measures Changing display, two kinds of pharmaceutical preparations are at the condition (IC of normal oxygen50, 25-50 M) under there is equivalent effect (Fig. 8).At anoxic bar Under part, MiaPaCa-2 cell is somewhat more sensitive (Fig. 8) to free 3-BrPA comparison β-CD-3-BrPA.That cultivates in 3D is thin Born of the same parents continue treated with medicaments, as described in example 4 above.Morphology, BLI, and immunofluorescence-base analysis confirmation 3-BrPA infiltration richness Matrix containing ECM and suppression cell proliferation, and the ability (Fig. 9-10) of inducing cell apoptosis, so, untreated " grape "-spline structure is bred and formed to MiaPaCa-2 cell at collagen 1 Medium Culture, and Suit-2 cell shows one more Invasive growth pattern, and visible cell projection (Figure 10) after the growth of 6 days.When processing with 3-BrPA, two kinds of cells The propagation of system is suppressed, with the cell process (Figure 10) substantially reducing in Suit-2 cell.Additionally, immunofluorescence imaging Confirm the Apoptosis induced by 3-BrPA dose-dependency.
In addition, 3-BrPA in Asia-the invasive ability of lethal drug control of the concentration pancreatic cancer cell uses matrigel to invade Attack determination method inspection.As shown in Figure 11 A-11B, local challenge MiaPaCa-2 cell and metastatic Suit-2 cell two Person is shown in aggressive during drug concentration as little as 12.5 M and reduces.In addition, the 3-BrPA of Asia-lethal dose is to Matrix-Metallo The effect that proteinase 9 (MMP-9) (a kind of mark for pancreatic cancer cell invasion and attack potentiality fully describing) is secreted, uses bright Glue zymography and Western blotting inspection (Jones etc. (1999)Annal. N. Y. Acad. Sci.880:288-307； Merdad etc. (2014)Anticanc. Res.34:1355-1366；Yang etc. (2001)J. Surg. Res. 98:33- 39).Therefore, substantially reducing of MMP-9 secretion is detected in two kinds of clone.This effect 3-BrPA of 6.25 M concentration Being initially observed, this concentration is the dosage of not inducing cell apoptosis or minimizing Cells viability, and at more metastatic Suit- Generation earlier in 2 clones (Figure 11 C-11D).
The system transmission of embodiment 6: β-CD-3-BrPA obtains strong internal anti-cancer effect
The anti-cancer effect of the β-CD-3-BrPA of system transmission uses the xenograft models of human pancreas cancer at nude mouse Middle inspection.Before selecting therapeutic dose for more detailed research, β-CD-3-BrPA and free both 3-BrPA is carried out non- The comparison dose escalating research of tumor animal.Therefore, after single injection, 20 mg/kg β-CD-3-BrPA and 10 mg/kg trip It is confirmed as half lethal dose (LD from 3-BrPA50), and when being administered systemically and every day 1 time, when continuing the course for the treatment of of 7 days, 5 mg/ Kg β-CD-3-BrPA is confirmed as and does not cause the safe dose of any toxicity.Then, altogether 42 have in situ-implant Accepted intraperitoneal (i.p.) injection β-CD-3-BrPA (N with the animal of the MiaPaCa-2 tumour of BLI-and USI-confirmation at random =21), gemcitabine (N=7), or β-CD (N=7).Another treated animal (N=7) with implant in situ is used free 3-BrPA treats.The animal treated by the free 3-BrPA (5 mg/kg are in 500 l salt solution) of intraperitoneal every day (i.p.) injection Show high treatment-xicity related and 3/7 (43%) animal before following up a case by regular visits to the collection of BLI for the first time dead (Figure 12 C). At the end of experiment (the 28th day), 2/7 (28%) is only had to remain (Figure 12 C) living with the animal of free drug treatment.This The death rate what remaining group in office of sample is not observed.(5 mg/kg are at 500 l salt for i.p. injection every day β-CD-3-BrPA In water) show strong anti-cancer effect, the impact (Figure 12 B) of the 14th day visible early stage after injecting for the first time.After treating 4 weeks, The BLI signal strength signal intensity carrying out between each group compares.Show, with the animal of β-CD control treatment, 140-times comparing with baseline to believe Number increase.The tumor growth that an appropriateness is slowed down, As time goes on, letter is observed in the animal of gemcitabine-treatment Number increase 60-times.The most important thing is, compare with gemcitabine and control group, demonstrate with the animal of β-CD-3-BrPA treatment Little or do not have signal progress (Figure 12).After reaching this terminal, put to death animal and collect tumour for further analysis. All animals are stood autopsy and gather in the crops organ (brain, the heart, lung, intestines, liver and kidney) for analyzing potential histologic lesion.With β- The animal of CD-3-BrPA treatment does not observes organ toxicity or tissue damage (Figure 12 D).Treated by β-CD-3-BrPA Oncological pathology analysis in animal shows very big tumor destruction, with the colliquative necrosis (Figure 13) of central area. The height of the Caspase-3 that the tumor region with intact cell connection shows cracking is expressed, and instruction fulminant tumour is thin Born of the same parents' apoptosis.Show substantially reducing of propagation with the animal of β-CD-3-BrPA treatment, as by the immunohistochemical difference of Ki67 (Figure 13) being evaluated by the mean of 17% and 51%.Additionally, compare with β-CD or gemcitabine group, with β-CD-3-BrPA treatment Animal show the relatively low expression of intra-tumor in treatment for MCT1 and GAPDH.
These results indicate, the β-CD-3-BrPA of system transmission realizes strong Anti-tumor effect in vivo, simultaneously when When comparing with free drug, under therapeutic dose, cause the toxicity of more much less.Additionally, the 3-BrPA of microencapsulation does not change medicine Resisting the effect of pancreatic cancer cell in vitro, this is used 2D under the conditions of normal oxygen and anoxic two kinds, and the 3D rich in ECM is thin Born of the same parents cultivate and are confirmed.3-BrPA suppresses the secretion of MMP-9 and the invasive energy of minimizing pancreatic cancer cell when sublethal dose Power also indicates the anti-metastatic potential of this medicine.
Being selectively targeting tumor metabolic is considered as preferably to treat option for a long time, but is not yet converted into clinical real Trample.The major limitation that use 3-BrPA reaches the milestone significance of system transmission capacity is reported owing to it is alkylated characteristic Toxicity (Ganapathy-Kanniappan and Geschwind (2013)Mol. Cancer12:152；Chang etc. (2007)Acad. Radiol. 14:85-92；Kunjithapatham etc. (2013)BMC Res. Not.6:277).As a result, locally Image-guided drug delivery has been explored as a kind of replacement therapy option；But, the actual application of these methods is limited to Treatment local disease (Ota etc. (2013)Target. Oncol.8:145-151；Geschwind etc. (2002)Canc. Res.62:3909-3913).Result described herein clearly illustrates medicine, when being suitably formulated for system transmission, is Extremely effective, thus the application by this compound is extended to actual any cancer.These results are with its Chinese traditional medicine only Other those results studied systematically being used for treating solid tumor with its free form are contrasted.In this study, swim Any significant tumor response (Cao etc. (2008) can not be caused from 3-BrPA when for the dosage of experiment described hereinClin. Canc. Res.14:1831-1839；Schaefer etc. (2012)Translat. Res. 159:51-57).Especially Ground, a research, it is explored free 3-BrPA and passes with system in subcutaneous pancreatic cancer xenografts for the HSP90 inhibitor combination Pass, when 3-BrPA is used alone (for security consideration, Per-Hop behavior is twice) with dosage 5 mg/kg, do not report any substantially Effect (Cao etc. (2008)Clin. Canc. Res.14:1831-1839).This unfavorable curative effect characteristic of free drug A possible explanation be 3-BrPA by the quick activation of the non-specific interaction with haemocyanin, this known as far back as System gives rear 2-3 minute and (Kunjithapatham etc. (2013) occurs in vivoBMC Res. Not.6:277).At this Observe some curative effects during a little dosage, but excessive toxicity, with the death to treatment-related in most animals, be Main result.It is not likely to be effective it is therefore believed that system gives free 3-BrPA and unwanted toxicity can be increased.Also phase Letter is in microencapsulated formulation, and 3-BrPA is more bioavailable for being absorbed into tumour cell, and mediates it to obvious The normal cell of toxicity is less available (Birsoy etc. (2013)Nature Genet.45:104-108；Zhang and Ma (2013) Advanc. Drug Deliv. Rev.65:1215-1233；Heidel and Schluep (2012)J. Drug Deliv. 2012:262731)。
One feature of pancreatic tumor tissue is the excessive buildup of intensive ECM, and this limits the diffusion of oxygen and creates one Be referred to as its significant chemoresistance and increase invasive height anoxic, irrigate bad tumor microenvironment (Yokoi and Fidler (2004) Clin. Canc. Res.10:2299-2306；Yang etc. (2001)J. Surg. Res. 98:33- 39).The research delivered confirms that the pancreatic tumor cell more than 30% is positioned at hypoxic tumor compartment, thus evades traditional Chemo-Therapy Treatment effect.These cells then proceed to re-form and a kind of become even more aggressive and the tumour to chemotherapy opposing (Guillaumond etc. (2013)Proc. Natl. Acad. Sci. U.S.A. 110:3919-3924).Described herein Result shows that 3-BrPA can block tumour glycolysis effectively, even if when it aggravates under anoxic conditions.On the contrary, have proven to Gemcitabine, even under maximum dose level, the also unable anoxic dealt with in pancreatic cancer cell.Up to the present, for thin in cancer The oxygen dependence of 3-BrPA in born of the same parents, conflicting data have been reported (Cao etc. (2008)Canc. Chemother. Pharmacol.62:985-994；Xiao etc. (2013)Oncol. Rep.29:329-334).But, there is 3-BrPA gram Take the important evidence (Xu etc. (2005) as the ability support of a drug-fast key mechanism for the anoxicCanc. Res. 65: 613-621).Especially, nearest research establishes the contact between the anoxic of MCT-1 and expression, and this is proved to be in anoxic Cell and tumor region over-express, is thus provided that for the function to the increase of the sensitiveness of 3-BrPA for the hypoxic tumor tissue Explain (Matsumoto etc. (2013)Magnet. Res. Med.69:1443-1450).It should be noted that in vitro Explore and combine gemcitabine and 3-BrPA, to be effectively realized the increase of curative effect；But, do not identify compound action, therefore, not Carry out respective experiment in vivo.
Additionally, use the 3D organotypic cell rich in collagen 1 to cultivate the model as the tumor microenvironment rich in ECM, Show to compare with monolayer cell culture, the energy of the minimizing without any measurable curative effect for the 3-BrPA successfully permeable matrices Power.3D cell culture model for research described herein is regarded as relative specificity, be primarily due to it be by Substrate composed, this matrix is simulated as at the ECM (Mollenhauer etc. rich in collagen 1 seen by mankind's vitro samples (1987) Pancreas2:14-24).Although for the purpose of drug test, the benefit of such external model is subject to more next More accreditations, these conditions in analogue body represent a bigger challenge (Longati etc. (2013)BMC Canc. 13: 95).When designing this research, different animal models to be considered.On the one hand, the original position heterograft being widely recognized as is used Thing model brings important advantage, for example repeatability, predictable tumor growth kinetics, and allows the gene of tumour cell Group modify, with expression specificity and imageable reporter gene (Kim etc. (2009)Nat. Protocol. 4: 1670- 1680).On the other hand, to reflect the degree of tumor microenvironment in human pathological unclear for these models.Although several definition Good mouse tumor model can more realistic simulation ECM-component and tumor hypoxia, these models seem to be poorly suited for standard Change the purpose (Guillaumond etc. (2013) of drug testProc. Natl. Acad. Sci. U.S.A. 110:3919- 3924).In terms of confirming the ability of 3-BrPA inhibitor cellular invasiveness in vitro, it is contemplated that metastatic Suit-2 heterograft The application of thing model.But, implant Suit-2 xenograft in situ and cause complication (that is, bloody ascites) and most animals Loss in 14 days after the implantation, this facilitates selection MiaPaCa-2 xenograft as a kind of feasible alternative.
One other afterclap is observed in the immunohistochemical analysis of the tumor tissues treated: and then The GAPDH as the molecular target of 3-BrPA that is expected and that be previously reported by exhausts, as the specific transport protein for 3-BrPA MCT-1 amount process sample in substantially reduce (Ganapathy-Kanniappan etc. (2012)Radiol. 262: 834-845).Up to now, the MCT-1 for the potential target as 3-BrPA exists, and does not has evidence.But, this breast Acid transporter albumen has been defined as the molecular target (Schneiderhan etc. (2009) of a suitable treatment of cancer repeatedlyGut 58:1391-1398；Shih etc. (2012)Oncotarget3:1401-1415；Sonveaux etc. (2012)PloS One 7: e33418)。
Therefore, result described herein determines that the 3-BrPA of microencapsulation can system transmission towards final realization as one The promising progress of the target of anti-glycolysis tumor therapy.The strong anti-cancer effect of β-CD-3-BrPA and favourable toxicity are special Levy the approach having laid the clinical testing led in suffering from the patient of pancreatic cancer and other potential malignant tumours.
By with reference to combination
All bibliography that the application quotes in the whole text, patent application, patent, and the content of the patent application announced, Yi Jitu Incorporated herein by reference with sequence table.Should be appreciated that, although many patent application, patent, and other are cited herein Bibliography, such reference is not intended that a part of accreditation forming this area common sense to these files any.
Equivalence
The experiment that it would be recognized by those skilled in the art that or use no more than routine can determine, invention described herein Many equivalence of particular.Such equivalence is intended to be covered by claims below.

Claims (30)

1. a composition, the medicine that it comprises cyclodextrin and below general formula represents:
Wherein, occur independently of one another:
X represents halide, sulphonic acid ester, carboxylate, alkoxide or amine oxide；
R1Expression OR, H, N (R ")2, C1-C6 alkyl, C6-C12 aryl, the miscellaneous alkyl of C1-C6 or C6-C12 heteroaryl；
R " represents H, C1-C6 alkyl or C6-C12 aryl；
R represents H, alkali metal, C1-C6 alkyl, C6-C12 aryl or C (O) R '；With
R ' represents H, C1-C20 alkyl or C6-C12 aryl,
The described medicine of wherein said cyclodextrin encapsulating.
2. the composition of claim 1, at least one hydroxyl of at least one α-D-glycopyranoside units of its cyclodextrin Chemical group is substituted by ionogenic chemical group.
3. the composition of claim 2, at least one hydroxy chemical of at least one α-D-glycopyranoside units wherein said Group is selected from C2, C3 and C6 hydroxy chemical group.
4. the composition of claim 3, C2, C3 and C6 of at least one α-D-glycopyranoside units of wherein said cyclodextrin Hydroxy chemical group is substituted by ionogenic chemical group.
5. the composition of any one of claim 1-4, at least one α-D-glycopyranoside units of wherein said cyclodextrin The 2nd, the 3rd, the 4th, the 5th, the 6th, the 7th, 8 and all of α-D-glycopyranoside units selected from described cyclodextrin.
6. the composition of any one of claim 1-5, wherein said ionogenic chemical group is on the position of all replacements It is identical.
7. the composition of any one of claim 1-6, wherein said ionogenic chemical group is alkalescent functional group or weak Acidic functionality.
8. the composition of claim 7, wherein alkalescent functional group (X) has according to CH3-X-Between 6.5 and 8.5 pKa。
9. the composition of claim 7, wherein weakly acidic functional group (Y) has according to CH3The pK between 4.0 and 6.5 of-Ya。
10. the composition of claim 7, wherein alkalescent or weakly acidic functional group are selected from amino, second diaminourea, dimethyl second two Amino, dimethyl benzene amino, dimethylnaphthalene amino, succinyl group, carboxyl, sulfonyl and Sulfuric acid functional groups.
The composition of any one of 11. claims 1-10, its cyclodextrin has the pK between 4.0 and 8.5a1。
The composition of any one of 12. claims 1-11, wherein composition is liquid or solid pharmaceutical preparation.
The composition of any one of 13. claims 1-12, its Chinese traditional medicine is uncharged or hydrophobic.
The liposome composition of any one of 14. claims 1-13, its cyclodextrin is selected from beta-schardinger dextrin, alpha-cyclodextrin, and Gamma-cyclodextrin.
The liposome composition of 15. claims 14, its cyclodextrin is beta-schardinger dextrin.
The composition of any one of 16. claims 1-15, its Chinese traditional medicine is the acid of 3-halogen acetone.
The composition of any one of 17. claims 1-16, its Chinese traditional medicine is 3-bromo acetone acid.
The composition of any one of 18. claims 1-17, wherein composition is formulated for system and gives.
The composition of any one of 19. claims 1-18, it also comprises anti-cancer therapeutic agent.
20. 1 kinds of kits, it comprises the composition of any one of claim 1-19, and operation instructions.
21. 1 kinds of treatments suffer from the method for the experimenter of cancer, and it includes that the right giving described subject's effective dose is wanted Seek the composition of any one of 1-20.
The method of 22. claims 21, wherein composition gives through system.
The method of 23. claims 22, wherein said system give selected from oral, intravenous, intraperitoneal, subcutaneous and intramuscular to Give.
The method of any one of 24. claims 21-23, wherein said experimenter is controlled by least one other anti-cancer therapy Treat.
The method of 25. claims 24, at least one other anti-cancer therapy wherein said is radiotherapy.
The method of any one of 26. claims 21-25, wherein said cancer is solid tumor.
The method of any one of 27. claims 21-26, wherein said cancer is selected from liver cancer, cancer of pancreas, lung cancer and breast cancer.
The method of 28. claims 27, wherein said cancer is liver cancer.
The method of any one of 29. claims 21-28, wherein said experimenter is mammal.
The method of 30. claims 29, wherein said mammal is people.
CN201580013842.3A 2014-01-14 2015-01-14 The cyclodextrin composite of the selective ATP inhibitor of encapsulating and application thereof Pending CN106102725A (en)

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